Improved rAAv vectors

ABSTRACT

Disclosed are methods for the use of therapeutic polypeptide-encoding polynucleotides in the creation of transformed host cells and transgenic animals. In particular, the use of recombinant adeno-associated viral (rAAV) vector compositions that specifically target mammalian cells, such as pancreatic islets cells, that express low-density lipoprotein receptors on their cell surface. The disclosed vectors comprise one or more polynucleotide sequences that express one or more mammalian polypeptides having therapeutic efficacy in the amelioration, treatment and/or prevention of AAT- or cytokine polypeptide deficiencies, such as for example in diabetes and related diseases, as well as a variety of autoimmune disorders including, for example, lupus and rheumatoid arthritis.

1. BACKGROUND OF THE INVENTION

The United States government has certain rights in the present inventionpursuant to grant numbers P50-HL59412, P01-NS36302, and P01-HL51811 fromthe National Institutes of Health.

1.1 FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to methods for usingrecombinant adeno-associated virus (rAAV) compositions that expressnucleic acid segments encoding therapeutic gene products in thetreatment of complex human disorders. In certain embodiments, theinvention concerns the use of rAAV in a variety of investigative,diagnostic and therapeutic regimens, including the treatment of diseasesof the pancreas and diabetes. Methods and compositions are also providedfor preparing rAAV-based vector constructs that target expression of oneor more therapeutic gene(s) to cells that express low-densitylipoprotein receptor on the cell surface, including liver, brain,muscle, and pancreatic islet cells, for use in a variety of viral-basedgene therapies, and in particular, treatment and/or prevention of humandiseases and disorders such as diabetes.

1.2 DESCRIPTION OF RELATED ART

1.2.1 Islet Cells

The pancreatic islets of Langerhans are critical for glucose homeostasisand their loss in Type I diabetes mellitus results in a disease thatgreatly increases the morbidity and mortality of affected individuals(Atkinson and Eisenbarth, 2001). Islet cell transplantation has providedan approach to the long-term remediation of the condition (Kenyon etal., 1998; Carroll et al., 1995; Ranuncoli et al., 2000). However, thecurrent paradigm of cadaveric donor-derived islet cell transplantationcreates a scenario in which allograft immunity compounds pre-existingauto-immunity leading to islet cell destruction. While certain newerimmunosuppressive protocols appear to be better tolerated (Shapiro etal., 2000), it would be highly desirable to enhance islet cellengraftment while decreasing immunosuppressive therapy. This couldpotentially be accomplished by genetically manipulating the islets toexpress anti-inflammatory cytokines or other mediators that could actlocally to decrease the immune response to the allograft and enhancecell viability (Tahara et al., 1992). Alternatively, insulin genetransfer into hepatocytes in vivo could provide an alternative source ofglucose-sensitive insulin release in insulin-deficient type I diabetes.

1.2.2 Deficiencies in the Prior Art

Currently, there are limited gene-therapy approaches to treatingdiseases of the liver and pancreas in an affected animal using rAAVvector-based gene therapies. Many such methods introduce undesirableside-effects, and do not overcome the problems associated withtraditional modalities and treatment regimens for such conditions. Also,limitations to the efficiency of rAAV serotype 2-mediated transfer havebeen reported for both the liver and the pancreas. Thus, the need existsfor improved rAAV expression systems that permit effective infection ofa broad range of cell types with the efficiency of transductionsufficient to provide therapeutic results. In particular, there is aneed for new rAAV-based vectors to facilitated improved methods fordelivery of polynucleotides that express selected therapeutic genes,antisense, and/or ribozymes to selected mammalian host cells thatexpress cell-sruface-localized lipoprotein. The availability of rAAVvectors and expression systems that provide modified capsids to mediatemore efficient transduction of liver and islet cells in particular aredesirable in the amelioration and treatment of many diseases anddysfunctions, including for example, diabetes, autoimmune disorders, andthe like. In particular, development of compositions and therapeuticmedicaments that comprise rAAV-based polynucleotide constructsspecifically targeted to cells that express low-density lipoproteinreceptors, including for example, the pancreatic islet cells of amammal, is particularly desirable.

2. SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations inherent inthe prior art by providing new rAAV-based genetic constructsspecifically targeted to mammalian cells, such as human liver, lung,muscle, and pancreatic islet cells that express one or more lipoproteinreceptor (LR) polypeptides on their cell surface. The improved rAAVvectors and expression systems of the present invention, as well as thevirions and viral particles that comprise them effectively mediate moreefficient transduction of selected mammalian cells, and particularlythose that express one or more low denity or very low densitylipoprotein receptors on their cell surface. The AAV vectors of thepresent invention comprise genetically-modified capsids that compriseone or more ligands that selectively targets lipoprotein receptors,including those found on certain liver, lung, muscle, and pancreaticislet cells.

The improved rAAV vectors and expression systems disclosed hereincomprise at least a first polynucleotide (or targeting region) thatencodes at least a first ligand that increases the affinity, binding,transduction of, or transfection of, selected mammalian cells thatexpress such LR's, including for example, low-density lipoproteinreceptors (LDLR) and very low-density lipoprotein receptors (VLDLR). Thenovel AAV-based expression systems and constructs of the invention alsofurther comprise at least a first polynucleotide that comprises anucleic acid segment that comprises at least a first promoter (and,optionally one or more enhancers) operably linked to a nucleic acidsegment that encodes one or more mammalian therapeutic peptides,polypeptides, ribozymes (catalytic RNA), or antisense nucleotides. Thedisclosed AAV-based expression systems may be comprised on a single AAVvector, which comprises both the targeting sequence and the therapeuticgene of interest, or optionally, may be comprised on two or morevectors, wherein the targeting sequence (preferably a peptide ligandthat has specificity for a mammalian LR) is comprised on one vector, andthe therapeutic construct is comprised on a second vector, such thatwhen the plurality of vectors are present within a population of AAVvirions, both the targeting sequence and the therapeutic gene sequenceare co-expressed to produce both the targeting ligand and thetherapeutic gene of interest.

In illustrative embodiments, the rAAV vectors of the present inventioncomprise at least a first nucleic acid segment that comprises at least afirst LR targeting sequence (such as for example a peptide ligandderived from a mammalian ApoE polypeptide) operably linked to a promoterthat expresses the sequence, and at least a second nucleic acid segmentthat comprises at least a first therapeutic gene operably linked to apromoter that expresses the gen

Such vectors are useful in the enhanced transfection of humanLR-expressing cells (including, but not limited to, for example, lung,liver, muscle, and pancreatic cells), for the prevention, treatment oramelioration of symptoms of one or more disorders, diseases,abnormalities, or dysfunctions of the cells, tissues, or organs thatcomprise the LR-expressing cells.

In particular, the invention provides genetic constructs encoding one ormore mammalian therapeutic peptides, polypeptides, ribozymes, orantisense nucleotides for use in therapy, such as in the amelioration,treatment and/prevention of such metabolic disorders as α₁-antitrypsindeficiency, and conditions such as diabetes and other dyfunctions of thepancreas, or pancreatic islet cells in particular.

Illustrative therapeutic agents include, for example, a polypeptideselected from those listed in Tables 14 and 15, and includebiologically-active, and/or therapeutically effective peptides,proteins, enzymes, antibodies, and antigen-binding fragments, includingfor example α₁-antitrypsin (AAT), growth factors, interleukins,interferons, anti-apoptosis factors, cytokines, anti-diabetic factors,anti-apoptosis agents, anti-tumor factors, and such like. When therapyof cancers or other hyperproliferative disorders are contemplated, theinvention contemplates the delivery of anti-cancer agents (such astoxins, tumor suppressors, and apoptosis agents). Likewise in thetreatment of certain other disorders, it may be desirable to provide oneor more therapeutic agents that inhibit, down-regulate, ablate, orotherwise kill selected cells that cause or contribute to the diseaseprocess.

Exemplary such therapeutic proteins include one or more polypeptidesselected from the group consisting of BDNF, CNTF, CSF, EGF, FGF, G-SCF,GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF,TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10,IL-11,IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18.

Exemplary therapeutic agents may also encompass one or morebiologically-active catalytic RNA molecules (ribozymes) that, whenintroduced into a host cell, selectively targets an mRNA sequence, andcleaves such sequence to prevent translation of substantial amounts ofthe selected mRNA into functional polypeptide. Such constructs areparticularly preferred in those diseases and dysfunctions that resultfrom the expression of mutant proteins, or from the over-expression ofone or more endogenous cellular polypeptides.

Exemplary therapeutic agents may also encompass one or morebiologically-active antisense oligonucleotides or polynucleotides that,when introduced into a selected mammalian host cell, selectivelyhybridize to an endogenous DNA or RNA sequence, and preventstranscription or translation of substantial amounts of the selected DNAor mRNA into functional RNA or polypeptide. Such constructs areparticularly preferred in those diseases and dysfunctions that resultfrom the expression of mutant genes or proteins, or from theover-expression of one or more endogenous cellular genes or polypeptidesencoded by them.

Exemplary therapeutic agents may also encompass one or morebiologically-active antibodies or antigen binding fragments that, whenintroduced into a selected mammalian host cell, selectively bind to,alter, or inactivate one or more endogenous peptides, polypeptides,proteins, or enzymes thus reducing, altering, or eliminating thebiological activity of the endogenous peptides, polypeptides, proteins,or enzymes. Such constructs are particularly preferred in those diseasesand dysfunctions that result from the expression of dysfunctional,deleterious, or biologically harmful polypeptides.

The invention also provides vectors, expression systems, virions, viralparticles, and compositions comprising them for use in the preparationof medicaments, and also methods for their use in preventin, treating orameliorating the symptoms of one or more deficiencies or dysfunctions ina mammal, such as for example, a polypeptide deficiency or polypeptideexcess in a mammal, and particularly for treating or reducing theseverity or extent of deficiency in a human manifesting one or more ofthe disorders linked to a deficiency in such polypeptides in cells andtissues of the human pancreas. In a general sense, the method involvesadministration of an rAAV-based genetic construct that specificallytargets LR-presenting cells, such as pancreatic islet cells, and thatencodes one or more therapeutic peptides, polypeptides, ribozymes, orantisense nucleotides, in a pharmaceutically-acceptable vehicle to theanimal in an amount and for a period of time sufficient to treat orameliorate the deficiency in the animal suspected of suffering from sucha disorder. In particular the invention contemplates the treatmentand/or prevention of diabetes and related disorders by specificallytargeting pancreatic islet cells with sufficient amounts of anrAAV-delivered therapeutic ribozyme-, antisense-, peptide- orpolypeptide-encoding nucleic acid segment.

In one embodiment, the invention provides an adeno-associated viralvector comprising at least a first polynucleotide that encodes atherapeutic peptide or polypeptide operably linked to a nucleic acidsegment that comprises an LR-targeting sequence and at least a firstpromoter capable of expressing the nucleic acid segment in a host celltransformed with such a vector to produce the encoded peptide orpolypeptide. In preferred embodiments, the nucleic acid segment encodesa mammalian, and in particular, a human therapeutic peptide orpolypeptide or a biologically active fragment or variant thereof. In onesuch embodiment, the therapeutic polypeptide is an AAT or cytokinepolypeptide. Alternatively, the therapeutic constructs of the inventionmay encode polypeptides of primate, simian, murine, porcine, bovine,equine, epine, canine, feline, ovine, caprine, or lupine origin. Inillustrative embodiments, the LR-targeting sequence is a peptidefragment of a mammalian ApoE polypeptide that has been show to enhancethe targeting of the rAAV construct to the LR-expressing pancreaticislet cells to provide therapeutic levels of the selected protein, e.g.,AAT or cytokine, to the transfected cells.

In another embodiment, the invention provides an adeno-associated viralvector comprising at least a first polynucleotide that encodes acatalytic RNA molecule, or ribozyme, operably linked to a nucleic acidsegment that comprises an LR-targeting sequence and at least a firstpromoter capable of expressing the nucleic acid segment in a host cellthat comprises the vector. In preferred embodiments, the nucleic acidsegment encodes a ribozyme sequence that specifically cleaves amammalian, and in particular, a human mRNA sequence such that theencoded polypeptide is reduced, or no longer expressed from the mRNA.Such constructs are particularly preferred when the therapeutic regimeninvolves eliminating, reducing, or affecting the expression of one ormore polynucleotides in a cell comprising the catalytic RNA-encodingsequence.

In a further embodiment, the invention provides a recombinantadeno-associated viral vector that comprises at least a firstpolynucleotide encoding an antisense molecule, operably linked to anucleic acid segment that comprises an LR-targeting sequence and atleast a first promoter capable of expressing the nucleic acid segment ina host cell that comprises the vector. In preferred embodiments, thenucleic acid segment encodes an antisense sequence that specificallybinds to, or inactivates, a mammalian, and in particular, a human mRNAsequence such that expression of the mRNA is altered, and as a result,the amount of the peptide or polypeptide normally produced fromtranslation of the mRNA is altered, reduced, or eliminated in a cellthat comprises the vector. Such constructs are particularly preferredwhen the therapeutic regimen involves eliminating, reducing, oraffecting the expression of one or more polynucleotides in a cell thatcomprises the rAAV that expresses the selected antisense molecule.

Another aspect of the invention concerns recombinant adeno-associatedviral vectors that comprise at least a first polynucleotide encoding anantibody or an antigen-binding fragment, operably linked to a nucleicacid segment that comprises an LR-targeting sequence and at least afirst promoter capable of expressing the nucleic acid segment in a hostcell that comprises the vector. In preferred embodiments, the nucleicacid segment encodes an antibody or an antigen-binding fragment thatspecifically binds to, alters, or inactivates, a mammalian, and inparticular, a human peptide or polypeptide such that the biologicalactivity of the peptide or polypeptide is altered as a result ofinteraction with the rAAV vector-encoded antibody or antigen-bindingfragment. Such constructs are particularly preferred when thetherapeutic regimen involves providing an antibody or an antigen-bindingfragment to a host cell to reduce, alter, or prevent the biologicalactivity of one or more peptides or polypeptides in a cell thatcomprises the rAAV that expresses the selected antibody orantigen-binding fragment.

In a further embodiment, the invention provides a recombinantadeno-associated viral vector that comprises at least a firstpolynucleotide encoding an epitopic peptide, operably linked to anucleic acid segment that comprises an LR-targeting sequence and atleast a first promoter capable of expressing the nucleic acid segment ina host cell that comprises the vector. In preferred embodiments, thenucleic acid segment encodes an epitopic peptide that specifically bindsto, alters, or inactivates, a mammalian, and in particular, a humanantibody or antigen-binding fragment, such that the biological activityof the antibody or antigen-binding fragment is altered, reduced, oreliminated, as a result of interaction with the rAAV vector-encodedepitopic peptide. Such constructs are particularly preferred when thetherapeutic regimen involves providing a small peptide epitope to a cellto alter or prevent the biological activity of one or more antibodies orantigen binding fragments present in a cell that comprises the rAAV thatexpresses the selected epitopic peptide.

Another aspect of the invention provides an improved recombinantadeno-associated viral vector that comprises at least a firstpolynucleotide comprising a first nucleic acid segment that encodes amodified AAV capsid protein that comprises at least one exogenous aminoacid sequence that binds to a mammalian lipoprotein receptor. While theinventors contemplate that almost any of the AAV capsid proteins may betargeted for inclusion of the exogenous LR targeting ligand (so long asthe essential functions of those capsid proteins are not impaired oreliminated), exemplary capsid proteins include, but are not limited toVp1, Vp2 or Vp3 capsid proteins.

In illustrative embodiments, the mammalian cells targeted by theseimproved AAV vectors include those mammalian (and preferably human)cells that include one or more low-density lipoprotein (LDL) or very lowdensity lipoprotein (VLDL) receptors on their cell surfaces.

The rAAV virions and viral particles of the present invention mayinclude any of the identified AAV serotypes, including, but not limitedto, rAAV serotype 1 (rAAV1), rAAV serotype 2 (rAAV2), rAAV serotype 3(rAAV3), rAAV serotype 4 (rAAV4) and rAAV serotype 5 (rAAV5) and rAAVserotype 6 (rAAV6), and such like.

The rAAV vector constructs of the invention preferably comprise at leasta first sequence that targets the construct to the cell membrane of amammalian pancreatic cell, and in particular, that targets the viralvector construct to cells that express lipoprotein receptorpolypeptides, and in particular LDLR or VLDLR polypeptides. Exemplarysuch tissues in the mammal include, for example, liver, brain, muscle,and pancreatic cells. In illustrative embodiments, a polynucleotidecomprising a segment that encodes a portion of the human ApoEpolypeptide was used to selectively target the expression of the encodedtherapeutic peptide, polypeptide, antisense, or ribozyme, to producetherapeutically-effective levels of the peptide, polypeptide, antisense,or ribozyme when suitable LR-expressing mammalian cells were providedwith the genetic construct.

The invention also provides a method for targeting an AAV virion orviral particle to a mammalian cell that comprises a cell-surfacelipoprotein receptor. The method generally involves at least the stepof: providing to a population of cells an AAV virion or viral particlethat comprises one or more of the disclosed rAAV vectors or rAAVexpression systems, in an amount and for a time effective to target thevirion or the viral particle to cells of the population that express acell-surface lipoprotein receptor.

The invention further provides a method for targeting an expressedtherapeutic agent to a mammalian cell that comprises a cell-surfacelipoprotein receptor. The method generally involves at least the step ofproviding to a mammal that comprises a population of such cells aneffective amount of one or more of the recombinant adeno-associatedviral expression systems disclosed herein.

Likewise, using one or more of the disclosed vectors or expressionsystems, the invention also provides methods for preventing, treating orameliorating the symptoms of a disease, dysfunction, or deficiency in aselected mammal in need of such treatment. These methods generallyinvolve at least the step of providing to or administering to the mammalone or more of the therapeutic rAAV virions, or plurality of viralparticles in an amount and for a time sufficient to treat or amelioratethe symptoms of the disease, dysfunction, or deficiency in the mammal.Such methods are contemplated to be particularly useful in the treatmentof human beings that have, are suspected of having, or diagnosed with,or at risk for developing one or more diseases, dysfunctions, orconditions in which the delivery of a therapeutic agent would bebeneficial in treating or preventing such conditions. The inventorscontemplate that owing to the surprising and remarkable efficiency atwhich the disclosed vectors target pancreatic islet cells, such methodswould be particularly beneficial to the treatment of pancreaticdisorders including, for example, diabetes, autoimmune disorders, orcancer.

In such methods, the virions or plurality of viral particles may beadministered to the mammal using conventional administration means, suchas, for example, intramuscularly, intravenously, subcutaneously,intrathecally, intraperitoneally, or by direct injection into an organor a tissue (including for example, the pancreas, liver, heart, lung,brain, kidney, joint, or muscle tissues).

The vector constructs and expression systems of the invention comprisinga sequence encoding an expressed therapeutic agent preferably compriseat least a first constitutive or inducible promoter, with promotersselected from the group consisting of a CMV promoter, a β-actinpromoter, an insulin promoter, a hybrid CMV promoter, a hybrid β-actinpromoter, a hybrid insulin promoter, an EF1 promoter, a U1a promoter, aU1b promoter, a Tet-inducible promoter and a VP16-LexA promoter beingparticularly useful in the practice of the invention. The promoters maybe homologous promoters, but may also encompass heterologous promotersthat are capable of directing expression of an operatively-linkedtherapeutic agent in a mammalian cell.

In exemplary embodiments, a polynucleotide encoding a therapeuticpolypeptide such as AAT, IL-4, IL-6, IL-10, IL-10 or viral IL-10(MacNeil et al., 1990; Go et al., 1990; Hsu et al., 1990; Moore et al.,1990) comprising an Ile to Ala mutation at amino acid (aa) position 87(IL-10[187A]) (see Ding et al., 2000), was placed under the control ofthe chicken β-actin promoter and used to produce therapeuticallyeffective levels of the encoded therapeutic polypeptide when suitablehost pancreatic islets cells were transformed with the rAAV geneticconstruct (FIG. 13).

The vector constructs of the present invention may also furtheroptionally comprise one or more native, synthetic, or hybrid regulatoryor “enhancer” elements, for example, a CMV enhancer, a syntheticenhancer, or a tissue- or cell-specific enhancer, such as for example, apancreatic-specific enhancer, a liver-specific enhancer, a lung-specificenhancer, a muscle-specific enhancer, a kidney-specific enhancer, or anislet cell-specific enhancer, or such like. Such elements are typicallypositioned upstream (or 5′) of the coding sequence, but alternatively,positioning downstream (or 3′) of the coding sequence may also beemployed in certain therapeutic constructs, so long as the enhancer orregulatory sequence employed is operably positioned within the constructso as to have an effect on transcription of the encoded therapeuticagent.

The vector constructs of the present invention may also furtheroptionally comprise one or more native, synthetic, or hybridpost-transcriptional regulatory elements that may function to helpstabilize the RNA and increase overall expression of the therapeuticpolypeptide. An exemplary such element is the woodchuck hepatitis viruspost-transcriptional regulatory element (WPRE) (see Paterna et al., 2000and Loeb et al., 1999).

The vectors may also further optionally comprise one or more intronsequences to facilitate improved expression of the therapeutic genesplaced under the control of the promoter and/or promoter/enhancerregulatory regions.

In illustrative embodiments, the invention concerns rAAV virions andviral particles that comprise a capsid protein such as Vp1 or Vp2modified to further comprise at least a first peptide ligand thattargets the virions and viral particles to a mammalian cell thatexpresses an LR on its cell surface. Such virions and particles areparticular desirable as vehicles for the delivery of genetic sequencesthat encode one or more therapeutic agents, including for examplebiologically-active peptides, polypeptides, antisenses, or ribozymes.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise oneor more of the vectors disclosed herein, such as for examplepharmaceutical formulations of the vectors intended for administrationto a mammal through suitable means, such as, by intramuscular,intravenous, or direct injection to one or both cells, tissues, organs,or organ systems of a selected mammal. Typically, such compositions willbe formulated with pharmaceutically-acceptable excipients as describedhereinbelow, and may comprise one or more liposomes, lipids, lipidcomplexes, microspheres or nanoparticle formulations to facilitateadministration to the selected organs, tissues, and cells for whichtherapy is desired.

The invention also encompasses recombinant host cells that comprise oneor more of the disclosed AAV vectors, virions, viral particles, or viralexpression systems.

Such cells are preferably mammalian host cells such as a pancreatic,kidney, muscle, liver, heart, lung, or brain cells. Particularlypreferred mammalian host cells are human pancreatic islet cells thatcomprise one or more of the improved AAV vectors disclosed herein.

Therapeutic kits for treating or ameliorating the symptoms of an AAT orinterleukin deficiency, including for example, diabetes or a relateddisorder of the pancreas also form important aspects of the presentinvention. Such kits typically comprise one or more of the disclosed AAVvectors, virions, virus particles, host cells, or compositions describedherein, and instructions for using the kit.

Another important aspect of the present invention concerns methods ofuse of the disclosed vectors, virions, compositions, and host cellsdescribed herein in the preparation of medicaments for treating orameliorating the symptoms of such a disease or dysfunction, or otherconditions resulting from an AAT or interleukin polypeptide deficiencycondition in a mammal. Such methods generally involve administration toa mammal, or human in need thereof, one or more of the disclosedvectors, virions, host cells, or compositions, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a deficiencyin the affected mammal. The methods may also encompass prophylactictreatment of animals suspected of having such conditions, oradministration of such compositions to those animals at risk fordeveloping such conditions either following diagnosis, or prior to theonset of symptoms. Such symptoms may include, but are not limited to,diabetes, rheumatoid arthritis, lupus, hyperinsulinemia,hypoinsulinemia, liver dysfunction, and a variety of autoimmunedisorders.

2.1 LR Targeting Peptide Sequences and Polypeptide Compositions

The present invention provides improved AAV constructs that express atleast a first mammalian LR targeting ligand or peptide integrated intoone of the three AAV capsid proteins, VP1, VP2, or VP3. Such constructspreferably include a sequence region in such modified capsid proteins,such that it further comprises the sequence of any one of SEQ IDNO:1-10, and preferably one of the sequences disclosed in SEQ ID NO:9 orSEQ ID NO:10. In certain embodiments, such constructs will morepreferably include a sequence region in such modified capsid proteins,such that it further comprises the sequence of any one of SEQ IDNO:11-20, and preferably one of the sequences disclosed in SEQ ID NO:19or SEQ ID NO:20. Such sequences may also further optionally comprise thesequence of SEQ ID NO:21, such that both peptide epitopes are expressedin one or more of the capsid proteins. In certain other embodiments,such constructs will even more preferably still include a sequenceregion in such modified capsid proteins, such that it further comprisesthe sequence of any one of SEQ ID NO:22-31, and preferably one of thesequences disclosed in SEQ ID NO:30 or SEQ ID NO:31.

In one embodiment, the invention provides AAV vectors that comprise atleast a first LR targeting ligand that comprises at least a firstisolated peptide of from 10 to about 60 amino acids in length, or atleast a first nucleic acid segment that encodes such a target peptide,wherein the peptide comprises a first contiguous amino acid sequenceaccording to any one of SEQ ID NO:1 to SEQ ID NO:31, and moreparticularly, a contiguous amino acid sequence according to any one ofSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:30, orSEQ ID NO:31, with peptides comprising one or more of the primary aminoacid sequences disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10 being particularly preferred.

The invention encompasses peptides that may be of any intermediatelength in the preferred ranges, such as for example, those peptides ofabout 60, about 55, about 50, about 45, about 40, about 35, about 30,about 25, about 20, or even about 15, 14, 13, 12, or 11 amino acids orso in length, as well as those peptides having intermediate lengthsincluding all integers within these ranges (e.g., the peptides may beabout 59, about 58, about 57, about 56, about 55, about 54, about 54,about 52, about 51, about 50, about 49, about 48, about 47, about 46,about 44, about 43, about 42, about 41, about 39, about 38, about 27, oreven about 36 or so amino acids in length, etc.). In particularembodiments, when smaller peptides are preferred, the length of thepeptide may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or even 20 or soamino acids in length, so long as the peptide comprises at least a firstcontiguous amino acid sequence as disclosed herein, such that thepeptide retains substantial LR binding activity. Likewise, when slightlylonger peptides are preferred, the length of the peptide may be about21, or about 22, or about 23, or about 24, or even about 25 or so aminoacids in length, so long as the peptide comprises at least a firstcontiguous amino acid sequence according to any one of the sequencesdisclosed herein, such that when expressed, the peptide retainssubstantial binding to mammalian cells that express one or mor LRs.

Likewise, the LR targeting peptides may be on the order of about 26, orabout 27, or about 28, or about 29, or about 30, or about 31, or about32, or about 33, or about 34, or even about 35 or so amino acids inlength,

Alternatively, the LR peptide may comprise an isolated peptide of from11 to about 60 amino acids in length, wherein the peptide comprises anamino acid sequence that consists of the sequence of any one of SEQ IDNO:1 to SEQ ID NO:31. Likewise, the LR targeting sequence may comprisean isolated peptide of from 12 to about 50 amino acids in length,wherein the peptide comprises an amino acid sequence that consists ofthe sequence of any one of SEQ ID NO:1 to SEQ ID NO:31. In fact, in somecircumstances it may be desirable that the LR targeting sequence encodedby the modified AAV vectors of the invention comprise an isolatedpeptide of from 13 to about 40 amino acids in length, wherein thepeptide comprises an amino acid sequence that consists of the sequenceof any one of SEQ ID NO:1 to SEQ ID NO:31. As such, isolated peptides offrom 14 to about 30 amino acids in length that comprise an amino acidsequence that consists of the sequence of any one of SEQ ID NO:1 to SEQID NO:31 are all within the scope of the present invention.

Preferred LR targeting peptides of the present invention likewiseencompass those from about 9 or 10 to about 55 or 60 amino acids inlength, those from 11 or 12 to about 45 amino or 50 acids in length, aswell as those from 13 or 14 to about 35 or 40 amino acids in length, andthose from 15 or 16 to about 30 amino acids in length. Likewise,preferred LR targeting ligands useful in targeting the AAV virions andviral particles of the present invention to a mammalian cell thatexpresses a cell-surface LR include those peptides from 16 to about 30amino acids in length, and any and all lengths, and sub-ranges oflengths within the overall preferred range of peptides of from 12 toabout 50 amino acids or so in length. In certain embodiments, theinvention may also encompass those LR targeting peptides having a lengthof about 8 or 9 amino acids in length, and that comprise essentially allof the sequence of any one of SEQ ID NO:1 to SEQ ID NO:10, so long asthe peptides retain substantial binding affinity for a mammalian LR.

Throughout this disclosure, a phrase such as “a sequence as disclosed inSEQ ID NO:1 to SEQ ID NO:10” is intended to encompass any and allcontiguous amino acid sequences disclosed by any of these sequenceidentifiers, and particularly, the peptide sequences disclosed in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. In fact, theinvention encompasses peptides and polynucleotides encoding them thatcomprise at least a first contiguous amino acid sequence as disclosed inany one of the sequences identified herein.

The AAV vectors of the invention also encompass those vectors thatcomprise at least a first DNA sequence that encodes an LR targetingligand that comprise a biologically-active molecule, and preferablythose peptides of from 10 to abut 60 or so amino acids in length thatcomprise, consist essentially of, or consist of, an amino acid sequencein any one of SEQ ID NO:1 to SEQ ID NO:31. As such, nucleotide sequencesthat encode a peptide that consists essentially of, or consists of, anamino acid sequence in any one of SEQ ID NO:1 to SEQ ID NO:31.

The invention also encompasses oligonucleotides and polynucleotides thatcomprise at least a first sequence region that encodes one or more ofthe LR targeting peptides or peptide variants as disclosed herein. Suchpolynucleotides may comprise a sequence region of 30 to about 300nucleotides in length, or a sequence region of 33 to about 270nucleotides in length, or a sequence region of 36 to about 240nucleotides in length, or a sequence region of 39 to about 210, or about180, or about 150, or about 120, or even about 90, 80, 70, or 60 or sonucleotides in length.

The peptides, polynucleotides, polypeptides, vectors, virus, and hostcells of the invention, as well as compositions comprising them mayfurther optionally comprise one or more detection reagents, one or moreadditional diagnostic reagents, one or more control reagents, and/or oneor more therapeutic reagents. In the case of diagnostic reagents, thecompositions may further optionally comprise one or more detectablelabels that may be used in both in vitro and/or in vivo diagnostic andtherapeutic methodologies. In the case of therapeutic compositions andformulations, the compositions of the invention may also furtheroptionally comprise one or more additional anti-cancer, or otherwisetherapeutically-beneficial components as may be required in particularcircumstances, and such like.

As noted above, the peptides of the present invention may comprise oneor more variants of the amino acid sequences as disclosed herein. An LRtargeting peptide “variant,” as used herein, is a peptide that differsfrom a particular LR targeting peptide primary amino acid sequence inone or more substitutions, deletions, additions and/or insertions, solong as the biological functional activity of the peptide (i.e., thepeptide's ability to bind to a mammalian LR, or the peptide's ability toselectively target an AAV capid to a cell that expresses such amammalian LR) is substantially retained (i.e., the ability of thevariant to bind to an LR is not substantially diminished relative to anative unmodified LR targeting peptide). In other words, the ability ofa peptide variant to bind to an LR may be enhanced or may be unchanged,relative to the peptide from which the LR targeting variant was derived.

Preferably, the biological activity of a peptide variant will not bediminished by more than 1%, and preferably still will not be diminishedby more than 2%, relative to the biological activity of the unmodifiedpeptide. More preferably, the biological activity of an LR targetingpeptide variant will not be diminished by more than 3%, and morepreferably still will not be diminished by more than 4%, 5%, 6%, 7%, 8%,or 9%, relative to the biological activity of the unmodified peptide.More preferably still, the biological activity of a peptide variant willnot be diminished by more than 10%, and more preferably still, will notbe diminished by more than 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,or 20% relative to the biological activity of the correspondingunmodified peptide.

Based upon % sequence homology, preferred peptide variant of the presentinvention include those peptides that are from 10 to about 60 aminoacids in length, and that comprise at least a first sequence region thatis at least 66% identical to at least one of the amino acid sequencesdisclosed in any one of SEQ ID NO:1 through SEQ ID NO:31, and morepreferably those that comprise at least a first sequence region that isat least 75% identical to at least one of the amino acid sequencesdisclosed in any one of SEQ ID NO:1 through SEQ ID NO:31. Morepreferably, based upon % sequence homology, preferred peptide variantsof the present invention are those peptides that comprise at least afirst sequence region that is at least 83% identical to at least one ofthe amino acid sequences disclosed in any one of SEQ ID NO:1 through SEQID NO:31, and more preferably those that comprise at least a firstsequence region that is at least 91% identical to at least one of theamino acid sequences disclosed in any one of SEQ ID NO:1 through SEQ IDNO:31.

Such peptide variants may typically be prepared by modifying one of thepeptide sequences disclosed herein, and particularly by modifying theprimary amino acid sequence of one or more of the LR targeting peptidesdisclosed in any one of SEQ ID NO:1 through SEQ ID NO:31. Thesebiological functional equivalent peptides may encompass primary aminoacid sequences that differ from the original peptide sequences disclosedin any one of SEQ ID NO:1 through SEQ ID NO:31 by one or moreconservative amino acid substitutions.

It has been found, within the context of the present invention, that arelatively small number of conservative or neutral substitutions (e.g.,1, 2, 3, or 4) may be made within the sequence of the LR targetingpeptides disclosed herein, without substantially altering the biologicalactivity of the peptide, or without substantially reducing the bindingof the peptide to a mammalian LR. Suitable substitutions may generallybe identified by using computer programs, as described hereinbelow, andthe effect of such substitutions may be confirmed based on the abilityof the modified peptide to compete with, for example, the peptide of SEQID NO:1 for binding to the human LR.

Accordingly, within certain preferred embodiments, a peptide for use inthe disclosed diagnostic and therapeutic methods may comprise a primaryamino acid sequence in which one or more amino acid residues aresubstituted by one or more replacement amino acids, such that theability of the modified peptide to compete with the peptide of SEQ IDNO:1 for binding to the human LR. is not significantly diminished oraltered.

As described above, LR targeting peptide variants are those that containone or more conservative substitutions. A “conservative substitution” isone in which an amino acid is substituted for another amino acid thathas similar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe peptide to be substantially unchanged. Amino acid substitutions maygenerally be made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values include leucine, isoleucine andvaline; glycine and alanine; asparagine and glutamine; and serine,threonine, phenylalanine and tyrosine. Examples of amino acidsubstitutions that represent a conservative change include: (1)replacement of one or more Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, orThr; residues with one or more residues from the same group; (2)replacement of one or more Cys, Ser, Tyr, or Thr residues with one ormore residues from the same group; (3) replacement of one or more Val,Ile, Leu, Met, Ala, or Phe residues with one or more residues from thesame group; (4) replacement of one or more Lys, Arg, or His residueswith one or more residues from the same group; and (5) replacement ofone or more Phe, Tyr, Trp, or His residues with one or more residuesfrom the same group.

A variant may also, or alternatively, contain nonconservative changes,for example, by substituting one of the amino acid residues from group(1) with an amino acid residue from group (2), group (3), group (4), orgroup (5). Variants may also (or alternatively) be modified by, forexample, the deletion or addition of amino acids that have minimalinfluence on the immunogenicity, secondary structure and hydropathicnature of the peptide.

2.2 Polynucleotide Compositions

The present invention concerns AAV polynucleotide constructs, vectors,and expression systems that encode one or more therapeutic peptides,polypeptides, antisense, or ribozymes, and that further encode at leasta first ligand that selectively targets AAV virions and virus particlesthat comprise such constructs, vectors, and expression systems to one ormore mammalian cells that express LR on their cell surface as describedherein. Such polynucleotides may be single-stranded (coding orantisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. Additional coding or non-coding sequencesmay, but need not, be present within a polynucleotide of the presentinvention, and a polynucleotide may, but need not, be linked to othermolecules and/or support materials.

The disclosed polynucleotides may encode native orsynthetically-modified peptides, proteins, antisense molecules, orribozymes, or may encode one or more biologically-active, ortherapeutically-effective variants thereof as described herein.Targeting sequence polynucleotide variants may contain one or moresubstitutions, additions, deletions and/or insertions such that theaffinity of the AAV virion for the cellular LR is not substantiallyaltered or diminished, relative to a native unmodified peptide ligandsequence. Preferred targeting peptide variants contain amino acidsubstitutions, deletions, insertions and/or additions at no more thanabout 4, about 3, about 2, or about 1 amino acid position within thesequence. When stated as a percentage, the modification will be no morethan about 30%, more preferably at no more than about 25% or about 20%,and more preferably still, at no more than about 15% or 10%, of theamino acid positions relative to the corresponding native unmodifiedamino acid sequence.

Likewise, polynucleotides encoding such peptide variants shouldpreferably contain nucleotide substitutions, deletions, insertionsand/or additions that change no more than about 4, about 3, about 2, orabout 1 of the triplets that encode the peptide targeting sequence. Whenstated as a percentage, the modification of the underlying DNA sequencethat encodes the targeting sequence preferably will not change more thanabout 25%, more preferably at no more than about 20% or 15%, and morepreferably still, at no more than about 10% or 5%, of the nucleotidepositions relative to the corresponding polynucleotide sequence thatencodes the native unmodified peptide sequence. Certain polynucleotidevariants, of course, may be substantially homologous to, orsubstantially identical to the corresponding region of the nucleotidesequence encoding an unmodified peptide. Such polynucleotide variantsare capable of hybridizing to a naturally occurring DNA sequenceencoding the selected sequence under moderately stringent, to highlystringent, to very highly stringent conditions.

Suitable moderately stringent conditions include pre-washing in asolution containing about 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0);hybridizing at a temperature of from about 50° C. to about 60° C. in5×SSC overnight; followed by washing twice at about 60 to 65° C. for 20min. with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS). Suitablehighly stringent conditions include pre-washing in a solution containingabout 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at atemperature of from about 60° C. to about 70° C. in 5×SSC overnight;followed by washing twice at about 65 to 70° C. for 20 min. with each of2×, 0.5× and 0.2×SSC containing 0.1% SDS). Representative examples ofvery highly stringent hybridization conditions may include, for example,pre-washing in a solution containing about 5×SSC, 0.5% SDS, 1.0 mM EDTA(pH 8.0); hybridizing at a temperature of from about 70° C. to about 75°C. in 5×SSC overnight; followed by washing twice at about 70° C. toabout 75° C. for 20 min. with each of 2×, 0.5× and 0.2×SSC containing0.1% SDS). Such hybridizing DNA sequences are also within the scope ofthis invention.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode an LR targeting peptide. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.

LR targeting peptide-encoding polynucleotides may also be synthesized byany method known in the art, including chemical synthesis (e.g., solidphase phosphoramidite chemical synthesis). Modifications in apolynucleotide sequence may also be introduced using standardmutagenesis techniques, such as oligonucleotide-directed site-specificmutagenesis (Adelman et al., 1983). Alternatively, RNA molecules may begenerated by in vitro or in vivo transcription of DNA sequences encodingan LR-targeting peptide, provided that the DNA is incorporated into avector with a suitable RNA polymerase promoter (such as T7 or SP6).Certain portions may be used to prepare an encoded peptide, as describedherein. In addition, or alternatively, a portion may be administered toa patient such that the encoded peptide is generated in vivo (e.g., bytransfecting antigen-presenting cells such as dendritic cells with acDNA construct encoding an LR targeting peptide, and administering thetransfected cells to the patient).

Polynucleotides that encode an LR targeting peptide may generally beused for production of the peptide, in vitro or in vivo. Polynucleotidesthat are complementary to a coding sequence (i.e., antisensepolynucleotides) may also be used as a probe or to inhibit thebiological activity (i.e., LR receptor binding activity) of the LRtargeting sequence. cDNA constructs that can be transcribed intoantisense RNA may also be introduced into cells of tissues to facilitatethe production of antisense RNA.

Any of the disclosed polynucleotides may be further modified to increasestability in vivo. The is particularly relevant when the therapeuticconstruct delivered by the disclosed AAV vectors is an antisensemolecular or a ribozyme. In such cases, possible modifications include,but are not limited to, the addition of flanking sequences at the 5′and/or 3′-ends; the use of phosphorothioate or 2′-o-methyl rather thanphosphodiesterase linkages in the backbone; and/or the inclusion ofnontraditional bases such as inosine, queosine and wybutosine, as wellas acetyl- methyl-, thio- and other modified forms of adenine, cytidine,guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety ofother nucleotide sequences using established recombinant DNA techniques.For example, a polynucleotide may be cloned into any of a variety ofcloning vectors, including plasmids, phagemids, lambda phage derivativesand cosmids. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.In general, a vector will contain an origin of replication functional inat least one organism, convenient restriction endonuclease sites and oneor more selectable markers. Other elements will depend upon the desireduse, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as topermit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed below. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vacciniaor other poxvirus (e.g., avian poxvirus). Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art.

2.3 Identification of Targeting Peptides that Bind to Mammalian LR

To identify LR targeting peptides useful in the creation of the AAVvectors of the present invention, one may employ a competitive bindingassay. Such assays are well-known to those of skill in the art, and mybe employed to quantitate the level of biological activity of candidateLR targeting ligands.

In conducting a competition binding study between a control LR targetingpeptide and any test peptide, one may first label the control (forexample, the peptide of SEQ ID NO:1) with a detectable label, such as,e.g., biotin or an enzymatic (or even radioactive) label to enablesubsequent identification. In these cases, one would pre-mix or incubatethe labeled control peptides with the test peptides to be examined atvarious ratios (e.g., 1:10, 1:100, or 1:1000, etc.) and (optionallyafter a suitable period of time) then assay the binding affinity of thelabeled control peptide ligand and compare this with a control value inwhich no potentially competing test peptide was included in theincubation.

The assay may again be any one of a range of peptide binding orcompetition assays based upon antibody hybridization, and the controlpeptides would be detected by means of detecting their label, e.g.,using streptavidin in the case of biotinylated antibodies or by using achromogenic substrate in connection with an enzymatic label (such as3,3′5,5′-tetramethylbenzidine (TMB) substrate with peroxidase enzyme) orby simply detecting a radioactive label. A peptide that binds to thesame LR as the labeled control ApoE peptide will be able to effectivelycompete for binding to LR and thus will significantly reduce controlpeptide ligand binding to LR, as evidenced by a reduction in boundlabel.

The reactivity of the (labeled) control LR targeting peptide in theabsence of a completely irrelevant peptide would be the control highvalue. The control low value would be obtained by incubating the labeledcontrol peptides with unlabelled peptides of exactly the same type, whencompetition would occur and reduce binding of the labeled peptides. In atest assay, a significant reduction in labeled peptide binding activityto LR in the presence of a test peptide is indicative of a testtargeting peptide that recognizes the same LR.

A significant reduction is a “reproducible”, i.e., consistentlyobserved, reduction in binding. A “significant reduction” in terms ofthe present application is defined as a reproducible reduction (in thecontrol peptide ligand (e.g., SEQ ID NO:1) binding to LR in an ELISA) ofat least about 70%, about 75% or about 80% at any ratio between about1:10 and about 1:100. Peptides with even more affinity for the LR willexhibit a reproducible reduction (in the binding of the label controlpeptide (e.g., SEQ ID NO:1) to LR in a suitable competitive bindingassay) of at least about 82%, about 85%, about 88%, about 90%, about 92%or about 95% or so at any ratio between about 1:10 and about 1:100.Complete or near-complete cross-blocking, such as exhibiting areproducible reduction in SEQ ID NO:1 binding to LR of about 99%, about98%, about 97% or about 96% or so, although by no means required topractice the invention, is certainly not excluded.

LR-targeting peptides that bind to substantially the same LR as thepeptide of SEQ ID NO:1 form other aspects of the invention.

In another embodiment, the invention provides a recombinantadeno-associated viral expression system that comprises: (a) a firstpolynucleotide comprising a first nucleic acid segment that encodes anAAV capsid protein that comprises an exogenous amino acid sequence thatbinds to a mammalian lipoprotein receptor; and (b) a secondpolynucleotide comprising a second nucleic acid segment that encodes anexpressed therapeutic agent.

The expressed therapeutic agent may be a peptide, polypeptide, ribozyme,or antisense molecule, and in certain preferred embodiments may be anenzyme, protein, or antibody. As in the case of the aforementionedvectors, the recombinant adeno-associated viral expression system of theinvention preferably comprise an exogenous amino acid sequence thatbinds to one or more mammalian LR polypeptides, such as the VLDL or LDLreceptors.

The expressed therapeutic agents of the invention will preferably bemammalian agents, such as those of human, primate, simian, murine,ursine, porcine, vulpine, bovine, feline, canine, ovine, equine, epine,caprine, or lupine origin.

In the recombinant adeno-associated viral expression systems of theinvention, the first and second polynucleotides may be comprised withina single rAAV vector, or they may each be comprised on distinct rAAVvectors: When present on separate vectors, the two vectors may beco-transfected to produce particles that comprise the genetic materialof both vectors, and thereby possess both altered capsid proteins thatinclude the LR targeting sequence, as well as the nucleic acid sequencethat encodes the selected therapeutic agent.

The invention also provides recombinant adeno-associated virus virions,and pluralities of such virions and viral particles, as well as hostcells, compositions, and kits that comprise one or more of the improvedrAAV vectors or rAAV expression systems disclosed herein.

2.4 Pharmaceutical Compositions

The genetic constructs of the present invention may be prepared in avariety of compositions, and may also be formulated in appropriatepharmaceutical vehicles for administration to human or animal subjects.The AAV molecules of the present invention and compositions comprisingthem provide new and useful therapeutics for the treatment, control, andamelioration of symptoms of a variety of disorders. Moreover,pharmaceutical compositions comprising one or more of the nucleic acidcompounds disclosed herein, provide significant advantages over existingconventional therapies—namely, (1) their reduced side effects, (2) theirincreased efficacy for prolonged periods of time, (3) their ability toincrease patient compliance due to their ability to provide therapeuticeffects following as little as a single administration of the selectedtherapeutic AAV composition to affected individuals. Exemplarypharmaceutical compostions and methods for their administration arediscussed in significant detail hereinbelow.

The invention also provides compositions comprising one or more of thedisclosed vectors, expression systems, virions, viral particles; ormammalian cells. As described hereinbelow, such compositions may furthercomprise a pharmaceutical excipient, buffer, or diluent, and may beformulated for administration to an animal, and particularly a humanbeing. Such compositions may further optionally comprise a liposome, alipid, a lipid complex, a microsphere, a microparticle, a nanosphere, ora nanoparticle, or may be otherwise formulated for administration to thecells, tissues, organs, or body of a mammal in need thereof. Suchcompositions may be formulated for use in therapy, such as for example,in the amelioration, prevention, or treatment of conditions such aspeptide deficiency, polypeptide deficiency, cancer, diabetes, autoimmunedisease, pancreatic disease, or liver disease or dysfunction.

Use of one or more of the disclosed compositions in the manufacture ofmedicaments for treating a variety of diseases is also an importantaspect of the invention. Such diseases include, for example, cancer,diabetes, cardiovascular diseases including coronary heart disease,angina, myocardial infarction, ischemias, restenosis, and strokes,atherosclerosis, pulmonary and circulatory diseases, including cysticfibrosis, hyperinsulinemia, hypoinsulinemia, adiposity, autoimmunediseases, lupus, inflammatory bowel disease, pancreatic dysfunction,hepatic dysfunction, biliary dysfunction and diseases, as well asneurological diseases including for example, Parkinson's, Alzheimer's,memory loss, and the like, as well as musculoskeletal diseasesincluding, for example, arthritis, ALS, MLS, MD, and such like, to nameonly a few.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thefollowing description taken in conjunction with the accompanyingdrawings, in which like reference numerals identify like elements, andin which:

FIG. 1 shows illustrative rAAV constructs of the invention. Linear mapsof the rAAV constructs used for ex vivo transduction of human and murineislets are depicted. ITR=AAV2 inverted terminal repeat,A_(n)=polyadenylation signal, luc-EYFP=translational fusion betweenfirefly luciferase and the enhanced yellow fluorescent protein, hAAT=thehuman α 1-antitrypsin coding sequence, EF=human elongation factor 1αpromoter, CMV=human cytomegalovirus immediate early enhancer/promoter, ahybrid construct with the CMV enhancer only followed by the chickenβ-actin promoter and a hybrid intron (upstream half of β-actin,downstream half of rabbit β-globin) is also shown.

FIG. 2 shows relative transcriptional activity of various promoters inhuman islets. Isolated human islets were transfected with rAAV proviralreporter constructs (CMV=CMV promoter luc-EYFP construct, CB=CMVenhancer/chicken β-actin construct, EF=human elongation factor 1αconstruct, Insulin=human insulin promoter construct) using Lipofectamine2000® (Gibco-BRL, Gaithersburg, Md.) either immediately after plating orafter treatment with tissue culture grade trypsin to loosen the isletcapsule. Luciferase assays were performed on cell lysates 48 hr aftertransfection and the values in relative light units (RLU) are indicatedon the y-axis. The bar heights indicate the mean of triplicate assays,the error bars depict the standard deviation of each set of values(*P<0.001, n=3).

FIG. 3 shows relative transduction efficiency of the same rAAV vectorpackaged into five different AAV serotypes in murine islets. TherAAV-CB-hAAT vector depicted in FIG. 1 was packaged into each of 5different AAV serotype capsids (shown on x-axis) and used to transducecultures of murine islets (at an MOI of 1×10⁴ particles per cell). Thelevel of expression of hAAT as determined by ELISA on supernatant mediataken 6 days after transduction is shown on the y-axis. The mean andstandard deviation of triplicate assays are shown in each instance.

FIG. 4 shows a diagram of the insertion site of a specific ligand forthe low-density lipoprotein receptor (LDL-R) into the AAV capsid resultsin enhanced targeting of pancreatic islet cells. The coding sequence ofthe three AAV capsid proteins, VP1, VP2, and VP3 are shown. The twoformer constructs represent N-terminal extensions of the latter. Theinsert at the amino acid+1 position of VP2 also appears within thecoding sequence of VP1. The human insulin promoter-driven luc-EYFPconstruct depicted in FIG. 1 was packaged into either wild-type (wt)AAV2 capsids or AAV2 with an additional insert consisting of the new IDLR ligand derived from ApoE. Confocal microscopy was performed 3 dayspost-transduction.

FIG. 5A and FIG. 5B show relative transduction efficiency of the samerAAV vector packaged into AAV2 and AAV2/ApoE serotypes in murine islets.The rAAV-CB-hAAT vector depicted in FIG. 1 was packaged into AAV2 orAAV2/ApoE capsids (shown on x-axis) and used to transduce cultures ofmurine islets. The level of expression of hAAT as determined by ELISA onsupernatant media taken 6 days (FIG. 5A) or 12 days (FIG. 5B) aftertransduction is shown on the y-axis. FIG. 5A and FIG. 5B show the levelsof expression at 6 days, and at 12 days after transduction,respectively.

FIG. 6 shows LDL-R targeting enhances gene transfer and expression afterportal vein injection. Aliquots of rAAV-CB-hAAT packaged into eithernative wild-type AAV2 capsid or into the capsid mutant displaying the28-amino acid ligand derived from ApoE were injected into the portalveins of cohorts of three C57B16 mice. The levels of hAAT present in thesera of these mice at 5 weeks after injection are shown.“PBS”=phosphate-buffered saline-injected control mice; “low AAV2”=doseof 7.5×10⁹ physical particles of vector in native AAV2 capsid; “highAAV2”=dose of 7.5×10¹⁰ physical particles of the native AAV2 vector;“low ApoE”=dose of 7.5×10⁹ particles of the LDL-R targeted mutant; “highApoE”=7.5×10¹⁰ particle dose of the latter vector.

FIG. 7A, FIG. 7B and FIG. 7C show AAV2-CMV-IL4 and IL10 constructs andexpression from these constructs after transfection into intact humanislet cells. FIG. 7A is a vector cassette map in which ITR=AAV invertedterminal repeat and CMVp=CMV immediate early promoter. The box followingthe promoter is the CMV first intron, and the box following the gene isthe SV40 polyA signal. FIG. 7B shows the concentration of IL-4 and IL-1048 hr after transduction of 0.2×10³ islets in a 35-mm well measured byantigen capture enzyme-linked immunosorbent assay (mean of threeexperiments, performed in duplicate). FIG. 7C shows the effect of rAAVtransduction on glucose-stimulated insulin release. AAV-CMV-IL4 andIL-10 constructs and expression from these constructs after transfectioninto human islet cells.

FIG. 8 shows the distribution of alanine scanning and HA epitopeinsertion mutants. Positions of the alanine scanning mutants (circles orsquares) and the HA insertion mutants (flagged circles or squares) areshown on a diagram of the putative secondary structure of the AAV capsidprotein adapted from a comparison of parvovirus capsid sequences byChapman and Rossman (1993). Some important amino acid positions andmutant positions are illustrated by numbers with short lines. Heavyarrows represent putative β sheets, and helices represent putative αhelices. The five putative loop regions are numbered I to V. Thephenotypes of the mutants are shown below: Mutated Class Mutant(s)Residues Primary phenotype Defect 1 mut1, mut2, mut3, mut9, 1, 2, 3, 9,11, 13, Wild type mut11, mut13, mut14, mut16, 14, 16, 17, 29, 32, mut17,mut29, mut32, mut38, 38, 43, 44, 45 mut 43, mut44, mut45 2a mut4, mut5,mut6, mut7, mut8, 4, 5, 6, 7, 8, 10, Partially defective mut10, mut12,mut15, mut18, 12, 15, 18, 30, mut30, mut34, mut36, mut48; 34, 36, 48,L1, L1, L3, L7, VPN1, VP1, L3, L7, VPN1, VPN2 VP1, VPN2 2b mut21, mut3921, 39 Partially defective Unstable capsid 2c mut41, L6 41, L6 Partiallydefective Heparan binding negative 3a mut26, mut27, mut28, mut33 26, 27,28, 33 Temperature sensitive 3b mut35 35 Temperature Heparan bindingsensitive negative 4a mut22, mut37; L5, L2 22, 37, L5, L2 Noninfectious4b mut19, mut20, mut23, mut24, 19, 20, 23, 24, Noninfectious No capsidmade mut25, mut42, mut46, mut47; 25, 42, 46, 47, VPN3, VPC VPN3, VPC 4cmut31 31 Noninfectious Empty capsid 4d mut40, L4 40, L4 NoninfectiousHeparan binding negative

FIG. 9 shows infectious titers of virus stocks containing wt and mutantcapsid proteins. The GFP fluorescent cell assay was used to titer virusstocks of wt and mutant virus stocks containing the pTRUF5 genome. 293cells were transfected with wt or mutant pIM45 complementing plasmid inthe presence of pTRUF5 and pXX6 at 39.5° C. and 32° C. Cells werecollected 48 hr posttransfection and then frozen and thawed three times.The crude lysate was used to infect 293 cells at 39.5° C. and 32° C.with Ad5 (MOI=10). The log value of the average infectious titer(infectious particles/milliliter) that was obtained from two independentstudies is shown. There was no significant difference between studies.The distribution of mutants unique to VP1, VP2, or VP3 is shown at thetop. Asterisks indicate temperature-sensitive mutants; noninfectiousmutants are indicated by check marks.

FIG. 10 shows infection of IB3 cells with wt and mutant virusescontaining a serpin ligand insertion. IB3 cells (1.5×10⁵ per 15-mm well)were infected with Ad5 for 60 min at an MOI of 10 and washed twice withmedium. The cells then were infected for 60 min at an MOI of 400 withrAAV containing a genome that expressed the hAAT gene under the controlof a CMV-β-actin hybrid promoter. Following infection, the cells werewashed with medium and incubated at 37° C. At 72-h postinfection, mediumsamples were taken to determine the AAT concentration by ELISA. Allstudies were done in triplicate, and the average for each study isshown. WT indicates that rAAV containing a wt AAV capsid (grown bycomplementation with pIM45) was used. VP1 virus was grown bycomplementation with a mutant plasmid containing the serpin ligandsequence (FVFLI) (SEQ ID NO:32) and DWLKAFYDKVAEDLDEAF (SEQ ID NO:21)substituted for the AAV capsid sequence after aa 34 of the cap ORF. VP2virus contained a serpin insertion (KFNKPFVFLI) (SEQ ID NO:33) at the Nterminus of VP2, aa 138 of the cap ORF. In the +HS samples, rAAVinfection was done in the present of soluble heparan sulfate at aconcentration of 2 mg/ml.

FIG. 11 shows ribbon diagrams of a dimer of the AAV VP3 model builtbased on structural alignments with the VP2 capsid protein of CPV. Theview is down an icosahedral twofold axis. The strands of the β-barrelmotif and the heparan binding region are shown. The open circlesidentify the locations of residue R432 mutated to an alanine in mut31.The filled circles identify the location of residues 266, 477, 591, and664 (which had HA insertions in mutants L1, L3, L6, and L7,respectively). The large triangle (dashed lines) indicates anicosahedral asymmetric unit. (For details, see FIG. 9, Wu et al.(2000)).

FIG. 12 illustrates luciferase reporter constructs to be used intransduction studies. ITR=AAV inverted terminal repeat; pA=bovine growthhormone polyadenylation signal, the various promoters are described inthe text. Each cassette is less than 4.5 kb.

FIG. 13 illustrates constructs of the invention using the IL-10(187A)mutated IL-10.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would nevertheless be a routineundertaking for those of ordinary skill in the art having the benefit ofthis disclosure.

4.1 RAAV-Mediated Transduction of Islet Cells

In the present invention, rAAV-mediated transduction has been enhancedby using alternative promoters, such as the human insulin promoter,alternative serotypes, and rAAV capsid mutants that incorporate a ligandderived from apolipoprotein E (ApoE) that is targeted to a cell surfacereceptor, such as the low density lipoprotein receptor (LDL-R) (Datta etal., 2000). The studies presented in the examples which follow indicatethat the transduction efficiency can be enhanced several thousand-fold,allowing for the use of MOIs as low as 5 i.u. per cell. The utility ofthe targeted AAV vectors has been demonstrated for in vivo transfer byportal vein injection, where a four-fold enhancement of transgeneexpression was observed. The improved rAAV vectors and expressionsystems described herein represent a significant advancement in the artof gene therapy, and provide methods of transducing LR-expressing cellswith substantially improved transduction frequencies. Such vectorsprovide an improved strategy for enhancing rAAV-mediated gene deliveryto other cells or tissues that are relatively non-permissive toinfection by wild-type AAV vectors.

To compare the islet cell transduction efficiency of various serotypesof AAV a CB-promoter-driven human α₁-antitrypsin (CB-hAAT) cassette wasutilized as a secreted reporter to transduce murine islets in culture.The level of hAAT expression achieved 6 days after transduction wassubstantially (2.5 times) higher with vector packaged in rAAV1 capsid ascompared with the rAAV2 serotype. rAAV3, rAAV4 and rAAV5 showed no hAATexpression. This is in contrast to human islets where rAAV5 showed aslight preference to rAAV2 transduction using the green fluorescentprotein (GFP) as a reporter.

The difference in transduction efficiency between serotypes suggeststhat receptor binding is a limiting step for transduction of islets. Inan effort to increase the transduction efficiency, the low-densitylipoprotein receptor (DLDR) on islets was targeted. A ligand derivedfrom ApoE (Datta et al., 2000; Perrey et al., 2001) was inserted into asite one residue downstream from the N-terminal methionine of VP2. SinceVP1 simply represents an N-terminal extension of VP2, this new peptideis displayed both within VP1 and VP2. Two different reporters werepackaged within the rAAV2-ApoE capsid, a human insulin promoter-drivenGFP (Ins-GFP) cassette and the same CB-hAAT cassette described above. Inthe GFP transduction studies, the ApoE capsid appeared substantiallymore efficient for islet cell transduction, with a greater number ofcells demonstrating native GFP fluorescence within each islet examined.

The enhancement of transduction was quantified in the hAAT expressionstudies. Equal volumes of CB-hAAT packaged into either wild-type AAV2capsid or AAV2-ApoE capsid were used to infect human islet cells and therelease of hAAT into the supematant medium was measured 72 hr later byELISA. The transduction efficiency was 90-fold greater (945 vs. 11ng/ml) with the ApoE insert. When the infectious titer of this vectorwas taken into account, however, the relative transduction efficiency interms of expression/MOI was approximately 9000-fold greater with theApoE capsid. This degree of enhancement is deduced since an equal volumeof a stock with a 100-fold lower infectious titer was used to generate90-fold greater hAAT expression. Even if one makes the most conservativeassessment of the enhancement factor, considering the physical titerrather than the infectious titer, the expression/particle was enhancedby 900-fold, since the ApoE stock had a physical particle titer of only10-fold lower than the wt-AAV stock. Taken together, these dataconvincingly demonstrate that receptor-targeting can greatly enhancerAAV transduction, regardless of the promoter or reporter gene systemused.

The ApoE capsid modification enables the improved virus to bind to thelow-density lipoprotein receptor (LDL-R). The LDL-R is found on manymore cell types than the normal receptor for AAV2 (heparan sulfateproteoglycan). Results have shown that the ApoE capsid modificationallows the rAAV to infect a wide variety of cell types with a muchhigher efficiency than the rAAV2 alone. This may also be very useful intreating diseases other than diabetes that require the transduction ofcell types that do not express high levels of the AAV2 receptor on theirsurface (e.g., liver, brain, muscle). By using the LDL-R to infect thesecells, it may also be possible to utilize these vectors in the treatmentof many other diseases.

rAAV vectors packaged with the ApoE capsid mutant are very useful notonly for the treatment of diabetes but many other diseases that requirerAAV to infect liver cells for sufficient therapy. Because liver cellscontain very high levels of LDL-R on the surface of the cells, the liveris another excellent target organ for gene delivery to produce asecreted protein. Brain, muscle, and other cells that express LDLRpolypeptides on the surface also benefit from the compositions of thepresent invention which provided selectively enhanced transduction ofcells bearing such surface receptor polypeptides.

Another important feature of the present invention is that much lowerdoses of vector are required to achieve the necessary levels of proteinexpression to correct the disease, because of the higher transductionefficiency with the ApoE capsid mutant packaged rAAV.

4.2 Auto-Immunity and Graft Rejection

The molecular immunology underlying auto-immunity and graft rejectionhas been extensively investigated in the context of islet cell andkidney transplantation in patients with type I diabetes mellitus. Thesestudies have yielded a number of candidate gene products that may extendgraft survival and/or prevent recurrent immune-mediated destruction of βcells. The selective manipulation of these genes could be safer and moreeffective than systemic pharmacological immune suppression. However, thepractical use of these gene products has been limited by the lack ofgene transfer vectors that are sufficiently safe, effective, andlong-lasting.

Over the past several years, recombinant adeno-associated virus (rAAV)vectors have been shown to be superior to other viral and non-viralsystems with regard to their in vivo safety, efficiency and duration ofaction both in animal models (Flotte et al., 1993; Conrad et al., 1996;Song et al., 1998; Kaplitt et al., 1994; Kessler et al., 1996) and inearly clinical trials (Wagner et al., 1998). In essence, this relates tothe intrinsic properties of wild-type AAV. Unlike adenoviruses andretroviruses, wild-type AAV naturally establishes persistent infectionsin humans (Berns and Linden, 1995) without any known pathology (Blacklowet al., 1971b; Blacklow, 1988) and with only modest immune responses(Beck et al., 1999; Hernandez et al., 1999). rAAV retains theseproperties and so has the potential to be an ideal vector for in vivogene transfer.

There are limitations to rAAV transduction, however, including themodest packaging capacity of the virion (approximately 5 kb). In somecell types, the efficiency of rAAV-mediated gene transfer has beenlimited by the abundance of either the attachment receptor (heparansulfate proteoglycan, HSP) or the co-receptors (fibroblast growth factorreceptor, FGF-R or β_(v)β₅-integrin) while expression is limited inother cell types due to transcriptional silencing (Summerford andSamulski, 1998; Summerford et al., 1999; Qing et al., 1999). It hasrecently been shown that the relative efficiency of transduction in onesuch cell type, the bronchial epithelial cell, can be enhanced bygenetic modification of the AAV capsid to include a small peptide ligandfor an alternative receptor (the serpin enzyme complex receptor, secR)(Wu et al., 2000). Studies indicate that islet cells are transduciblewith rAAV but that they require a high multiplicity of infection. It ishypothesized that this high dosage requirement indicates a relativescarcity of high affinity rAAV receptors on the cell surface.

4.3 Adeno-Associated Virus

Adeno-associated virus (AAV) is a parvovirus with a 4.7 kbsingle-stranded DNA genome (Carter et al., 1975; Muzyczka et al., 1984).It was discovered as a laboratory contaminant of adenovirus cultures(Atchison et al., 1966; Hoggan et al., 1966) and was subsequently foundto require adenovirus or another helper virus to replicate under mostcircumstances (Hoggan et al., 1968). AAV serotypes 1-6 are found inprimates, and AAV2 and 3 are particularly common in humans (Blacklow etal., 1967; Blacklow et al., 1968a; Blacklow et al., 1971a). AAV2 wasfound to be a frequent isolate among children experiencing an outbreakof adenovirus-induced diarrhea (Blacklow et al., 1968b). None of the AAVserotypes has ever been associated with any human disease (Flotte andCarter, 1995.

The AAV life cycle is quite unusual (Berns and Linden, 1995). AAV bindsto cells via a heparan sulfate proteoglycan receptor (Summerford andSamulski, 1998). Once attached, AAV entry is dependent upon the presenceof a co-receptor, which may consist of either the fibroblast growthfactor receptor (FGF-R) (Qing et al., 1999) or the α_(v)-β₅ integrinmolecule (Summerford et al., 1999). Cells infected with AAV and a helpervirus (or another adjunctive agent, such as UV irradiation or treatmentwith genotoxic agents) will undergo productive replication of AAV priorto cell lysis, which is induced by the helper rather than by AAV. Humancells infected with AAV alone, however, will become persistentlyinfected (Berns et al., 1975). This latency pathway often results incolinear integration of AAV sequences within the host cell genome(Cheung et al., 1980), often within a specific site on human chromosome19, the AAVS1 site (Kotin et al., 1990; Kotin et al., 1991; Kotin etal., 1992; Samulski et al., 1991; Samulski, 1993. While this site is notstrictly homologous to AAV, it contains consensus elements required forbinding and nicking by the AAV Rep protein, a non-structural proteinthat is also involved in productive replication and in transcriptionalregulation of the virus (Weitzman et al., 1994; Giraud et al., 1994;Giraud et al., 1995; Linden et al., 1996). Once AAV is integrated, itwill remain stable within infected cells for prolonged periods of time,up to 100 passages (Hoggan et al., 1972). Episomal forms of the virusmay also be present for extended periods in some circumstances (Afioneet al., 1996; Kearns et al., 1996; Flotte, 1994). If latently infectedcells are subsequently infected with a helper virus, the genome will beexcised and productive AAV replication and packaging will ensue(Senapathy et al., 1984; Afione et al., 1996).

The AAV genome consists of two 145-nucleotide inverted terminal repeat(ITR) sequences, each an identical palindrome at either terminus of thevirus, flanking the two AAV genes, rep and cap (Tratschin et al., 1984).The rep gene is transcribed from two promoters, the p5 promoter (at mapposition 5) and the p19 promoter (map position 19), which is embeddedwithin the coding sequence of the longer Rep proteins. In each case,both the spliced and unspliced transcripts are processed and translated.This allows for the production of 4 Rep proteins, Rep78, Rep68, Rep52,and Rep40. Rep78 and Rep68 are multifunctional DNA binding proteinswhich possess helicase activity and site-specific, strand-specificnickase activity, both of which are required for terminal resolution ofreplicating AAV genomes (Im and Muzyczka, 1990). The long Rep proteinsare also capable of binding to the chromosomal target sequence for AAVintegration, the AAVS1 site, and these proteins are required for normalintegration into this site. Finally, Rep78/68 are potent bi-functionaltranscription regulators that generally activate transcription from AAVpromoters during productive infection and repress their transcriptionduring latent infection (Pereira and Muzyczka, 1997; Pereira et al.,1997). The shorter Rep proteins, Rep52 and Rep40 act as modifierproteins for long Rep transcriptional activities, and are required forsequestration of single-stranded AAV genomes into capsids duringproductive infection.

The AAV cap gene is transcribed from the p40 promoter. The 5′ end of theMRNA transcript from p40 contains an intron which can utilize either oftwo downstream splice acceptor sites. The longer of the two processedmessages contains an ATG codon which is used in the translation of VP1,the longest of the three AAV capsid proteins. The shorter mRNA caninitiate translation using either a non-canonical ACG start codon, whichrepresents the start of VP2, or an ATG codon further downstream, whichcomprises the N-terminal Met of VP3 (Trempe and Carter, 1988). VP3 isthe shortest and most abundant of the AAV capsid proteins, but all threeare required for the production of infectious virions.

4.4 Recombinant AAV Vectors

Recombinant AAV (rAAV) vectors have been developed by replacement of theviral coding sequences with transgene of interest (Tratschin et al.,1984; Hermonat and Muzyczka, 1984. The ITR sequences must be retained inrAAV since these serve as origins for viral DNA replication and containthe packaging signals. The maximum packaging capacity of rAAV isapproximately 5 kb, including the ITRs, the transgene, its promoter, andpolyadenylation signal (Flotte et al., 1992; Dong et al., 1996). Thefull exploitation of rAAV for gene transfer has been limited in the pastprimarily by the packaging and purification process. In particular,contamination of rAAV vector preparations with wild-type AAV has beenfound to alter the biological behavior of the vector, and limitations onthe titers and infectivity of the vectors have limited their widespreaduse on the past. Recent advances in the packaging and purificationtechnology have resulted in a dramatic improvement in the expressionlevels that have been achievable in vivo. In particular, the use ofadenoviral plasmids and of complementing rep gene expression constructsthat express less of the longer Rep proteins (Rep68/78) has resulted ina substantial improvement in the efficiency of vector production on aper cell basis (Xiao et al., 1998; Li et al., 1997). The availability ofpackaging cell lines has also resulted in a substantial improvement inthe scale-ability of the packaging process (Clark et al., 1996; Flotteet al., 1995; Gao et al., 1998). Finally, the availability of severalcolumn chromatography methods, including heparin sulfate affinity columnchromatography, has allowed for the elimination of CsCl banding, whichin turn appears to have enhanced the infectivity of output particles(Zolotukhin et al., 1999).

rAAV vectors are uniquely suitable for in vivo gene therapy for geneticand metabolic disorders, since they are non-toxic (Flotte et al., 1993;Conrad et al., 1996; Flotte and Carter, 1998), highly efficient whenused at high titers, relatively non-immunogenic (Jooss et al., 1998;Hernandez et al., 1999; Beck et al., 1999), and very stable in theirpattern of expression. The utility of rAAV vectors for in vitro and invivo gene transfer has now been well established. There appear to beimportant tissue specific differences in rAAV effects, however. rAAVvectors have been found to be particularly efficient for gene transferinto terminally differentiated cells such as neurons (Kaplitt et al.,1994; McCown et al., 1996; Peel et al., 1997; Mandel et al., 1997),myofibers (Xiao et al., 1996; Kessler et al., 1996; Clark et al., 1997;Fisher et al., 1997; Song et al., 1998, and photoreceptor cells(Flannery et al., 1997; Lewin et al., 1998; Zolotukhin et al., 1996;Rolling et al., 1999). Gene transfer to the bronchial epithelium hasalso been demonstrated (Flotte et al., 1993; Conrad et al., 1996; Afioneet al., 1996; Flotte et al., 1998; Halbert et al., 1998, although theefficiency of transduction remains relatively low. Likewise, rAAVtransduction of hepatocytes has also been studied, and has been found tobe efficient enough to provide a potential therapeutic strategy forhemophilia B, by providing persistent and therapeutic concentrations ofhuman factor IX in mice (Snyder et al., 1997. However, in that study, insitu hybridization results indicated that only 5% of hepatocytes hadbeen transduced (Miao et al., 1998).

In the case of each of these two cell types, recent evidence has shownthat the efficiency can be enhanced by altering the capsid toincorporate ligands for a receptor that is abundantly expressed on thecell surface and by optimizing the promoter usage (Wu et al., 2000;Virella-Lowell et al., 1999). Similar manipulations are alsoadvantageous in pancreatic islet cells. Recent reports of severedose-related clinical adverse events due to adenovirus, although notdirectly reflective of rAAV, underscore the necessity of minimizing thedose of vector whenever possible.

4.5 Promoters and Enhancers

Recombinant AAV vectors form important aspects of the present invention.The term “expression vector or construct” means any type of geneticconstruct containing a nucleic acid in which part or all of the nucleicacid encoding sequence is capable of being transcribed. In preferredembodiments, expression only includes transcription of the nucleic acid,for example, to generate a biologically-active AAT or interleukinpolypeptide product from a transcribed gene.

Particularly useful vectors are contemplated to be those vectors inwhich the nucleic acid segment to be transcribed is positioned under thetranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrases “operatively positioned,” “undercontrol” or “under transcriptional control” means that the promoter isin the correct location and orientation in relation to the nucleic acidto control RNA polymerase initiation and expression of the gene.

In preferred embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a biologically-active AAT orinterleukin gene in its natural environment. Such promoters may includepromoters normally associated with other genes, and/or promotersisolated from any bacterial, viral, eukaryotic, or mammalian cell.

Naturally, it will be important to employ a promoter that effectivelydirects the expression of the biologically-active AAT orinterleukin-encoding DNA segment in the cell type, organism, or evenanimal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al.(1989), incorporated herein by reference. The promoters employed may beconstitutive, or inducible, and can be used under the appropriateconditions to direct high-level expression of the introduced DNAsegment.

At least one module in a promoter functions to position the start sitefor RNA synthesis. The best-known example of this is the TATA box, butin some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have beenshown to contain functional elements downstream of the start site aswell. The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter that-is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the biologically-active AAT or interleukinpolypeptide-encoding nucleic acid segment in the targeted cell. Thus,where a human cell is targeted, it is preferable to position the nucleicacid coding region adjacent to and under the control of a promoter thatis capable of being expressed in a human cell. Generally speaking, sucha promoter might include either a human or viral promoter, such as abeta-actin, CMV, an HSV promoter, or even a human insulin or otherpancreas-specific or otherwise inducible promoter. In certain aspects ofthe invention, the chicken beta-actin promoter has been demonstrated tobe particularly desirable in some embodiments disclosed herein.

In various other embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters that are well known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose. Tables 1 and 2 below listseveral elements/promoters that may be employed, in the context of thepresent invention, to regulate the expression of the presentbiologically-active AAT or interleukin polypeptide-encoding nucleic acidsegments comprised within the AAV vectors of the present invention. Thislist is not intended to be exhaustive of all the possible elementsinvolved in the promotion of transgene expression, but merely to beexemplary thereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression. Use ofa T3, T7 or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct. TABLE 1 PROMOTER AND ENHANCER ELEMENTSPROMOTER/ENHANCER REFERENCES Immunoglobulin Heavy Chain Banerji et al.,1983; Gilles et al., 1983; Grosschedl and Baltimore, 1985; Atchinson andPerry, 1986, 1987; Imler et al., 1987; Weinberger et al., 1984;Kiledjian et al., 1988; Porton et al; 1990 Immunoglobulin Light ChainQueen and Baltimore, 1983; Picard and Schaffner, 1984 T-Cell ReceptorLuria et al., 1987; Winoto and Baltimore, 1989; Redondo et al.; 1990 HLADQ a and DQ β Sullivan and Peterlin, 1987 β-Interferon Goodbourn et al.,1986; Fujita et al., 1987; Goodbourn and Maniatis, 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase Jaynes et al., 1988; Horlick and Benfield, 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein Karin et al., 1987; Culotta andHamer, 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminGene Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere and Tilghman, 1989 t-Globin Bodine andLey, 1987; Perez-Stable and Constantini, 1990 β-Globin Trudel andConstantini, 1987 e-fos Cohen et al., 1987 c-HA-ras Triesman, 1986;Deschamps et al., 1985 Insulin Edlund et al., 1985 Neural Cell AdhesionMolecule Hirsh et al., 1990 (NCAM) α_(1-Antitrypain) Latimer et al.,1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse or Type I Collagen Ripeet al., 1989 Glucose-Regulated Proteins (GRP94 Chang et al., 1989 andGRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A(SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey et al., 1989Platelet-Derived Growth Factor Pech et al., 1989 Duchenne MuscularDystrophy Klamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al.,1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr andClarke, 1986; Imbra and Karin, 1986; Kadesch and Berg, 1986; Wang andCalame, 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber and Lehman, 1975; Vasseur et al., 1980;Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983;de Villiers et al., 1984; Hen et al., 1986; Satake et al., 1988;Campbell and Villarreal, 1988 Retroviruses Kriegler and Botchan, 1982,1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988;Bosze et al., 1986; Miksicek et al., 1986; Celander and Haseltine, 1987;Thiesen et al., 1988; Celander et al., 1988; Chol et al., 1988; Reismanand Rotter, 1989 Papilloma Virus Campo et al., 1983; Lusky et al., 1983;Spandidos and Wilkie, 1983; Spalholz et al., 1985; Lusky and Botchan,1986; Cripe et al., 1987; Gloss et al., 1987; Hirochika et al., 1987;Stephens and Hentschel, 1987 Hepatitis B Virus Bulla and Siddiqui, 1986;Jameel and Siddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and Lee,1988; Vannice and Levinson, 1988 Human Immunodeficiency Virus Muesing etal., 1987; Hauber and Cullan, 1988; Jakobovits et al., 1988; Feng andHolland, 1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al.,1989; Laspia et al., 1989; Sharp and Marciniak, 1989; Braddock et al.,1989 Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foeckingand Hofstetter, 1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987;Quinn et al., 1989

TABLE 2 INDUCIBLE ELEMENTS ELEMENT INDUCER REFERENCES MT II PhorbolEster Palmiter et al., 1982; Haslinger (TFA) and Karin, 1985; Searle etal., Heavy metals 1985; Stuart et al., 1985; Imagawa et al., 1987, Karinet al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouseGlucocorticoids Huang et al., 1981; Lee et al., mammary 1981; Majors andVarmus, 1983; tumor virus) Chandler et al., 1983; Lee et al., 1984;Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI) × poly(rc)Tavernier et al., 1983 Adenovirus 5 E2 Ela Imperiale and Nevins, 1984Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin PhorbolEster (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al.,1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2- IL-6 Kunz et al., 1989 MacroglobulinVimentin Serum Rittling et al., 1989 MHC Class I Interferon Blanar etal., 1989 Gene H-2κb HSP70 Ela, SV40 Large Taylor et al., 1989; Taylorand T Antigen Kingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacqand Linzer, 1989 Tumor Necrosis FMA Hensel et al., 1989 Factor ThyroidThyroid Hormone Chatterjee et al., 1989 Stimulating Hormone a Gene

As used herein, the terms “engineered” and “recombinant” cells areintended to refer to a cell into which an exogenous DNA segment, such asDNA segment that leads to the transcription of a biologically-active AATor interleukin polypeptide or a ribozyme specific for such abiologically-active AAT or interleukin polypeptide product, has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells, which do not contain a recombinantlyintroduced exogenous DNA segment. Engineered cells are thus cells havingDNA segment introduced through the hand of man.

To express a biologically-active AAT or interleukin encoding gene inaccordance with the present invention one would prepare an rAAVexpression vector that comprises a biologically-active AAT orinterleukin polypeptide-encoding nucleic acid segment under the controlof one or more promoters. To bring a sequence “under the control of” apromoter, one positions the 5′ end of the transcription initiation siteof the transcriptional reading frame generally between about 1 and about50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The“upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded polypeptide. This is the meaning of“recombinant expression” in this context. Particularly preferredrecombinant vector constructs are those that comprise an rAAV vector.Such vectors are described in detail herein.

4.6 Ribozymes

In certain embodiments, the invention concerns the delivery oftherapeutic catalytic RNA molecules, or ribozymes, to selected mammaliancells.

Although proteins traditionally have been used for catalysis of nucleicacids, another class of macromolecules has emerged as useful in thisendeavor. Ribozymes are RNA-protein complexes that cleave nucleic acidsin a site-specific fashion. Ribozymes have specific catalytic domainsthat possess endonuclease activity (Kim and Cech, 1987; Gerlach et al.,1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein by reference) reports that certainribozymes can act as endonucleases with a sequence specificity greaterthan that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

Six basic varieties of naturally occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992).Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al.(1990) and U. S. Pat. No. 5,631,359 (specifically incorporated herein byreference). An example of the hepatitis δ virus motif is described byPerrotta and Been (1992); an example of the RNaseP motif is described byGuerrier-Takada et al. (1983); Neurospora VS RNA ribozyme motif isdescribed by Collins (Saville and Collins, 1990; Saville and Collins,1991; Collins and Olive, 1993); and an example of the Group I intron isdescribed in U. S. Pat. No. 4,987,071 (specifically incorporated hereinby reference). All that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

In certain embodiments, it may be important to produce enzymaticcleaving agents that exhibit a high degree of specificity for the RNA ofa desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required,although in preferred embodiments the ribozymes are expressed from DNAor RNA vectors that are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may also be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl.Publ. No. WO 94/02595, both hereby incorporated by reference; Ohkawa etal., 1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595 (each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure, as described herein.Those ribozymes with unfavorable intramolecular interactions between thebinding arms and the catalytic core are eliminated from consideration.Varying binding arm lengths can be chosen to optimize activity.Generally, at least 5 or so bases on each arm are able to bind to, orotherwise interact with, the target RNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe et al.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically>98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-flouro, 2′-o-methyl, 2′-H(for a review see e.g., Usman and Cedergren, 1992). Ribozymes may bepurified by gel electrophoresis using general methods or byhigh-pressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al., 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

A preferred means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells(Kashani-Sabet et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yuet al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993).Although incorporation of the present ribozyme constructs intoadeno-associated viral vectors is preferred, such transcription unitscan be incorporated into a variety of vectors for introduction intomammalian cells, including but not restricted to, plasmid DNA vectors,other viral DNA vectors (such as adenovirus vectors), or viral RNAvectors (such as retroviral, semliki forest virus, sindbis virusvectors).

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describesgeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraocular, retinal, subretinal,intraperitoneal and/or intrathecal delivery. More detailed descriptionsof ribozyme and rAAV vector delivery and administration are provided inInt. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO93/23569, each specifically incorporated herein by reference.

Ribozymes and the AAV vectored-constructs of the present invention maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of one or more retinaldiseases and/or disorders. In this manner, other genetic targets may bedefined as important mediators of the disease. These studies lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple ribozymes targeted to differentgenes, ribozymes coupled with known small molecule inhibitors, orintermittent treatment with combinations of ribozymes and/or otherchemical or biological molecules).

4.7 Ribozymes

In certain embodiments, the disclosed AAV constructs may be used todeliver one or more therapeutic antisense molecules to selectedmammalian cells. In the specification and claims, the letters, A, G, C,T, and U respectively indicate nucleotides in which the nucleoside isAdenosine (Ade), Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), andUridine (Ura). As used in the specification and claims, compounds thatare “antisense” to a selected sequence are those that have a nucleosidesequence that is complementary to the selected sense strand. Table 3shows the four possible sense strand nucleosides and their complementspresent in an antisense compound. TABLE 3 Sense Antisense Ade Thy GuaCyt Cyt Gua Thy Ade Ura Ade

The antisense compounds may have some or all of the phosphates in thenucleotides replaced by phosphorothioates (X=S) or methylphosphonates(X=CH₃) or other C₁₋₄ alkylphosphonates. The antisense compoundsoptionally may be further differentiated from native DNA by replacingone or both of the free hydroxy groups of the antisense molecule withC₁₋₄ aikoxy groups (R=C₁₋₄ alkoxy). As used herein, C₁₋₄ alkyl means abranched or unbranched hydrocarbon having 1 to 4 carbon-atoms.

The disclosed antisense compounds also may be substituted at the 3′and/or 5′ ends by a substituted acridine derivative. As used herein,“substituted acridine,” means any acridine derivative capable ofintercalating nucleotide strands such as DNA. Preferred substitutedacridines are 2-methoxy-6-chloro-9-pentylaminoacridine,N-(6-chloro-2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-3-aminopropanol,andN-(6-chloro-2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-5-aminopentanol.Other suitable acridine derivatives are readily apparent to personsskilled in the art. Additionally, as used herein “P(0)(0)-substitutedacridine” means a phosphate covalently linked to a substitute acridine.

As used herein, the term “nucleotides” includes nucleotides in which thephosphate moiety is replaced by phosphorothioate or alkylphosphonate andthe nucleotides may be substituted by substituted acridines.

The antisense compounds may differ from native DNA by the modificationof the phosphodiester backbone to extend the life of the antisense ON.For example, the phosphates can be replaced by phosphorothioates. Theends of the molecule may also be optimally substituted by an acridinederivative that intercalates nucleotide strands of DNA. Intl. Pat. Appl.Publ. No. WO 98/13526 and U.S. Pat. No. 5,849,902 (each specificallyincorporated herein by reference) describe a method of preparing threecomponent chimeric antisense compositions, and discuss many of thecurrently available methodologies for synthesis of substitutedoligonucleotides having improved antisense characteristics and/orhalf-life.

4.8 Peptide Nucleic Acid Compositions

In certain embodiments, the inventors contemplate the use of peptidenucleic acids (PNAS) in the practice of the methods of the invention.PNA is a DNA mimic in which the nucleobases are attached to apseudopeptide backbone (Good and Nielsen, 1997). PNAs may be utilized ina number of methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Anexcellent review of PNA including methods of making, characteristics of,and methods of using, is provided by Corey (1997) and is incorporatedherein by reference. As such, in certain embodiments, one may preparePNA sequences that are complementary to one or more portions of theβ₁-adrenoceptor-specific mRNA sequence, and such PNA compositions may beused to regulate, alter, decrease, or reduce the translation ofβ₁-adrenoceptor-specific mRNA, and thereby alter the level ofβ₁-adrenoceptor polypeptide in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., 1993; Hanvey et al.,1992; Hyrup and Nielsen, 1996; Nielsen, 1995). This chemistry has threeimportant consequences: firstly, in contrast to DNA or phosphorothioateoligonucleotides, PNAs are neutral molecules; secondly, PNAs areachiral, which avoids the need to develop a stereoselective synthesis;and thirdly, PNA synthesis uses standard Boc (Dueholm et al., 1992) orFmoc (Bonham et al., 1995) protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used (Christensen et al., 1995).

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass., USA). PNA syntheses by eitherBoc or Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., 1995).

4.9 Mutagenesis and Preparation of Modified Nucleotide Compositions

In certain embodiments, it may be desirable to prepared modifiednucleotide compositions, such as, for example, in the generation of thenucleic acid segments that encode either the peptide targeting ligand,and/or the therapeutic gene delivered by the disclosed rAAV vectors.Various means exist in the art, and are routinely employed by theartisan to generate modified nucleotide compositions.

Site-specific mutagenesis is a technique useful in the preparation andtesting of sequence variants by introducing one or more nucleotidesequence changes into the DNA. Site-specific mutagenesis allows theproduction of mutants through the use of specific oligonucleotidesequences which encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed.Typically, a primer of about 17 to 25 nucleotides in length ispreferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. As will be appreciated, the technique typically employs abacteriophage vector that exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage. These phage vectors arecommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis, which eliminates the step of transferringthe gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector that includes within its sequence a DNA sequence encoding thedesired ribozyme or other nucleic acid construct. An oligonucleotideprimer bearing the desired mutated sequence is synthetically prepared.This primer is then annealed with the single-stranded DNA preparation,and subjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected that include recombinant vectors bearing the mutated sequencearrangement.

The preparation of sequence variants of the selected nucleic acidsequences using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

4.10 Nucleic Acid Amplication

In certain embodiments, it may be necessary to employ one or morenucleic acid amplification techniques to produce the nucleic acidsegments of the present invention. Varioius methods are well-known toartisans in the field, including for example, those techniques describedherein:

Nucleic acid, used as a template for amplification, may be isolated fromcells contained in the biological sample according to standardmethodologies (Sambrook et al., 1989). The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a complementary DNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to the ribozymes or conserved flanking regions arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer”, as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with oneor more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (e.g. Affymax technology).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of thebest-known amplification methods is the polymerase chain reaction(referred to as PCR™), which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159 (each of which is incorporated hereinby reference in its entirety).

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR# amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal. (1989). Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin Int. Pat. Appl. Publ. No. WO 90/07641 (specifically incorporatedherein by reference). Polymerase chain reaction methodologies are wellknown in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, and incorporated herein by reference inits entirety. In LCR, two complementary probe pairs are prepared, and inthe presence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qβ Replicase (QβR), described in Int. Pat. Appl. No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolymerase will copy the replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA), described in U.S. Pat. Nos.5,455,166, 5,648,211, 5,712,124 and 5,744,311, each incorporated hereinby reference, is another method of carrying out isothermal amplificationof nucleic acids which involves multiple rounds of strand displacementand synthesis, i.e., nick translation. A similar method, called RepairChain Reaction (RCR), involves annealing several probes throughout aregion targeted for amplification, followed by a repair reaction inwhich only two of the four bases are present. The other two bases can beadded as biotinylated derivatives for easy detection. A similar approachis used in SDA. Target specific sequences can also be detected using acyclic probe reaction (CPR). In CPR, a probe having 3′ and 5′ sequencesof non-specific DNA and a middle sequence of specific RNA is hybridizedto DNA that is present in a sample. Upon hybridization, the reaction istreated with RNase H, and the products of the probe identified asdistinctive products that are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated.

Still another amplification methods described in GB Application No. 2202 328, and in Int. Pat. Appl. No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (FAS), including nucleic acid sequence basedamplification (NASBA) and 3SR Gingeras et al., Int. Pat. Appl. Publ. No.WO 88/10315, incorporated herein by reference. In NASBA, the nucleicacids can be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer that has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double-strandedDNA molecules are then multiply transcribed by an RNA polymerase such asT7 or SP6. In an isothermal cyclic reaction, the RNA's are reversetranscribed into single stranded DNA, which is then converted to doublestranded DNA, and then transcribed once again with an RNA polymerasesuch as T7 or SP6. The resulting products, whether truncated orcomplete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., Int. Pat. Appl. Publ. No. WO 89/06700 (incorporatedherein by reference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, ie., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR™” (Frohman, 1990, specifically incorporated herein by reference).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (see e.g., Sambrook et al., 1989).

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols. See Sambrook et al., 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

4.11 Methods of Nucleic Acid Delivery and DNA Tranfection

In certain embodiments, it is contemplated that one or more RNA, DNA,PNAs and/or substituted polynucleotide compositions disclosed hereinwill be used to transfect an appropriate host cell. Technology forintroduction of PNAs, RNAs, and DNAs into cells is well known to thoseof skill in the art.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Wong and Neumann, 1982; Fromm et al., 1985;Tur-Kaspa et al., 1986; Potter et al., 1984; Suzuki et al., 1998;Vanbever et al., 1998), direct microinjection (Capecchi, 1980; Harlandand Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;Fraley et al., 1979; Takakura, 1998) and lipofectamine-DNA complexes,cell sonication (Fechheimer et al., 1987), gene bombardment using highvelocity microprojectiles (Yang et al., 1990; Klein et al., 1992), andreceptor-mediated transfection (Curiel et al., 1991; Wagner et al.,1992; Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

Moreover, the use of viral vectors (Lu et al., 1993; Eglitis andAnderson, 1988; Eglitis et al., 1988), including retroviruses,baculoviruses, adenoviruses, adenoassociated viruses, vaccinia viruses,Herpes viruses, and the like are well-known in the art, and aredescribed in detail herein.

4.12 Expression Vectors

The present invention contemplates a variety of AAV-based expressionsystems, and vectors. In certain embodiments the preferred AAVexpression system comprises a nucleic acid segment that encodes atherapeutic antisense molecule. In another embodiment, a promoter isoperatively linked to a sequence region that encodes a functional mRNA,a tRNA, a ribozyme or an antisense RNA.

As used herein, the term “operatively linked” means that a promoter isconnected to a functional RNA in such a way that the transcription ofthat functional RNA is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a functional RNA are well known inthe art.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depend directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the functional RNA to which itis operatively linked.

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive terinih or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

4.13 Biological Functional Equivalents

Modification and changes to the structure of the polynucleotides andpolypeptides of wild-type rAAV vectors to provide the improved rAAVvirions as described in the present invention to obtain functional viralvectors that possess desirable characteristics, particularly withrespect to improved delivery of therapeutic gene constructs to selectedmammalian cell, tissues, and organs for the treatment, prevention, andprophylaxis of various diseases and disorders, as well as means for theamelioration of symptoms of such diseases, and to facilitate theexpression of exogenous therapeutic and/or prophylactic polypeptides ofinterest via rAAV vector-mediated gene therapy. As mentioned above, oneof the key aspects of the present invention is the creation of one ormore mutations into specific polynucleotide sequences that encode one ormore of the rAAV capsid proteins. In certain circumstances, theresulting encoded capsid polypeptide sequence is altered by thesemutations, or in other cases, the sequence of the polypeptide isunchanged by one or more mutations in the encoding polynucleotide toproduce modified vectors with improved properties for effecting genetherapy in mammalian systems.

When it is desirable to alter the amino acid sequence of a polypeptideto create an equivalent, or even an improved, second-generationmolecule, the amino acid changes may be achieved by changing one or moreof the codons of the encoding DNA sequence, according to Table 4.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the polynucleotide sequences disclosed herein,without appreciable loss of their biological utility or activity. TABLE4 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGCUGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, ie. still obtain abiological functionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No.4,554,101, incorporated herein by reference, states that the greatestlocal average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take several of theforegoing characteristics into consideration are well known to those ofsklll in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

4.14 Pharmaceutical Compositions

In certain embodiments, the present invention concerns formulation ofone or more of the rAAV compositions disclosed herein inpharmaceutically acceptable solutions for administration to a cell or ananimal, either alone or in combination with one or more other modalitiesof therapy, and in particular, for therapy of the mammalian pancreas andtissues thereof, such as for example, islet cells.

It will also be understood that, if desired, nucleic acid segments, RNA,DNA or PNA compositions that express one or more of thebiologically-active AAT or interleukin therapeutic gene products asdisclosed herein may be administered in combination with other agents aswell, such as, e.g., proteins or polypeptides or variouspharmaceutically-active agents, including one or more systemic or directadministrations of AAT or interleukin polypeptides, or biologicallyactive fragments, or variants thereof. In fact, there is virtually nolimit to other components that may also be included, given that theadditional agents do not cause a significant adverse effect upon contactwith the target cells or host tissues. The rAAV compositions may thus bedelivered along with various other agents as required in the particularinstance. Such compositions may be purified from host cells or otherbiological sources, or alternatively may be chemically synthesized asdescribed herein. Likewise, such compositions may further comprisesubstituted or derivatized RNA, DNA, or PNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the AAVvector-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraopancreatically, parenterally, intravenously, intramuscularly,intrathecally, or even orally, intraperitoneally, or by nasalinhalation, including those modalities as described in U.S. Pat. Nos.5,543,158; 5,641,515 and 5,399,363 (each specifically incorporatedherein by reference in its entirety). Solutions of the active compoundsas freebase or pharmacologically acceptable salts may be prepared insterile water and may also suitably mixed with one or more surfactants,such as hydroxypropylcellulose. Dispersions may also be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activeAAV vector-delivered biologically-active AAT or interleukinpolypeptide-encoding polynucleotides in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

The AAV vector compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human, and in particular, whenadministered to human cells that express LDLR polypeptides. Thepreparation of an aqueous composition that contains a protein as anactive ingredient is well understood in the art. Typically, suchcompositions are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection can also be prepared. The preparation can alsobe emulsified.

4.15 Liposme-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the rAAV vectordelivered gene therapy compositions of the present invention may beformulated for delivery either encapsulated in a lipid particle, aliposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Pat. No.5,741,516, specifically incorporated herein by reference in itsentirety). Further, various methods of liposome and liposome likepreparations as potential drug carriers have been reviewed (Takakura,1998; Chandran et al., 1997; Margalit, 1995; U.S. Pat. Nos. 5,567,434;5,552,157; 5,565,213; 5,738,868 and 5,795,587, each specificallyincorporated herein by reference in its entirety).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs(Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989;Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses(Faller and Baltimore, 1984), transcription factors and allostericeffectors (Nicolau and Gersonde, 1979) into a variety of cultured celllines and animals. In addition, several successful clinical trailsexamining the effectiveness of liposome-mediated drug delivery have beencompleted (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier etal., 1988). Furthermore, several studies suggest that the use ofliposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mori and Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for h or days, depending on theircomposition, and half lives in the blood range from min to several h.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically acceptablenanocapsule formulations of the AAV vector-based polynucleotidecompositions of the present invention. Nanocapsules can generally entrapcompounds in a stable and reproducible way (Henry-Michelland et al,1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoidside effects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for use inthe present invention. Such particles may be are easily made, asdescribed (Couvreur et al., 1980; Couvreur, 1988; zur Muhlen et al.,1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat.No. 5,145,684, specifically incorporated herein by reference in itsentirety).

4.16 Additional Modes of Delivery

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe disclosed rAAV vector based polynucleotide compositions to a targetcell or animal. Sonophoresis (ie., ultrasound) has been used anddescribed in U.S. Pat. No. 5,656,016 (specifically incorporated hereinby reference in its entirety) as a device for enhancing the rate andefficacy of drug permeation into and through the circulatory system.Other drug delivery alternatives contemplated are intraosseous injection(U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898),ophthalmic formulations (Bourlais et al., 1998), transdermal matrices(U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlleddelivery (U.S. Pat. No. 5,697,899), each specifically incorporatedherein by reference in its entirety.

4.17 Therapeutic and Diagnostic Kits

The invention also encompasses one or more disclosed rAAV compositionstogether with one or more pharmaceutically-acceptable excipients,carriers, diluents, adjuvants, and/or other components, as may beemployed in the formulation of particular rAAV-polynucleotide deliveryformulations, and in the preparation of therapeutic agents foradministration to a mammal, and in particularly, to a human, for one ormore of the conditions described herein. In particular, such kits maycomprise one or more of the disclosed rAAV compositions in combinationwith instructions for using the viral vector in the treatment of suchdisorders in a mammal, and may typically further include containersprepared for convenient commercial packaging.

As such, preferred animals for administration of the pharmaceuticalcompositions disclosed herein include mammals, and particularly humans.Other preferred animals include primates, simians, murines, bovines,ovines, lupines, vulpines, equines, porcines, canines, and felines aswell as any other mammalian species commonly considered pets, livestock,or commercially relevant animal species. The composition may includepartially or significantly purified rAAV compositions, either alone, orin combination with one or more additional active ingredients, which maybe obtained from natural or recombinant sources, or which may beobtainable naturally or either chemically synthesized, or alternativelyproduced in vitro from recombinant host cells expressing DNA segmentsencoding such additional active ingredients.

Therapeutic kits may also be prepared that comprise at least one of thecompositions disclosed herein and instructions for using the compositionas a therapeutic agent. The container means for such kits may typicallycomprise at least one vial, test tube, flask, bottle, syringe or othercontainer means, into which the disclosed rAAV composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticcomposition is also provided, the kit may also contain a second distinctcontainer means into which this second composition may be placed.Alternatively, the plurality of biologically-active therapeuticcompositions may be prepared in a single pharmaceutical composition, andmay be packaged in a single container means, such as a vial, flask,syringe, bottle, or other suitable single container means. The kits ofthe present invention will also typically include a means for containingthe vial(s) in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desiredvial(s) are retained. 4.18 Exemplary Definitions

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including and not limited to genomic or extragenomicDNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but notlimited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleicacid segments either obtained from native sources, chemicallysynthesized, modified, or otherwise prepared in whole or in part by thehand of man.

Unless defmed otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defmed below:

A, an: In accordance with long standing patent law convention, the words“a” and “an” when used in this application, including the claims,denotes “one or more”.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a polynucleotide such as astructural gene to synthesize the encoded peptide or polypeptide.

Promoter: a term used to generally describe the region or regions of anucleic acid sequence that regulates transcription.

Regulatory Element: a term used to generally describe the region orregions of a nucleic acid sequence that regulates transcription.

Structural gene: A gene or sequence region that is expressed to producean encoded peptide or polypeptide.

Transformation: A process of introducing an exogenous polynucleotidesequence (e.g., a vector, a recombinant DNA or RNA molecule) into a hostcell or protoplast in which that exogenous nucleic acid segment isincorporated into at least a first chromosome or is capable ofautonomous replication within the transformed host cell. Transfection,electroporation, and naked nucleic acid uptake all represent examples oftechniques used to transform a host cell with one or morepolynucleotides.

Transformed cell: A host cell whose nucleic acid complement has beenaltered by the introduction of one or more exogenous polynucleotidesinto that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell, or from the progeny or offspring ofany generation of such a transformed host cell.

Vector: A nucleic acid molecule, typically comprised of DNA, capable ofreplication in a host cell and/or to which another nucleic acid segmentcan be operatively linked so as to bring about replication of theattached segment. A plasmid, cosmid, or a virus is an exemplary vector.

The terms “substantially corresponds to”, “substantially homologous”, or“substantial identity” as used herein denotes a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared. The percentage of sequence identity may be calculated over theentire length of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides. Desirably, which highly homologous fragments aredesired, the extent of percent identity between the two sequences willbe at least about 80%, preferably at least about 85%, and morepreferably about 90% or 95% or higher, as readily determined by one ormore of the sequence comparison algorithms well-known to those of skillin the art, such as e.g., the FASTA program analysis described byPearson and Lipman (1988).

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by the hand of man in alaboratory is naturally-occurring. As used herein, laboratory strains ofrodents that may have been selectively bred according to classicalgenetics are considered naturally occurring animals.

As used herein, a “heterologous” is defined in relation to apredetermined referenced gene sequence. For example, with respect to astructural gene sequence, a heterologous promoter is defined as apromoter which does not naturally occur adjacent to the referencedstructural gene, but which is positioned by laboratory manipulation.Likewise, a heterologous gene or nucleic acid segment is defined as agene or segment that does not naturally occur adjacent to the referencedpromoter and/or enhancer elements.

“Transcriptional regulatory element” refers to a polynucleotide sequencethat activates transcription alone or in combination with one or moreother nucleic acid sequences. A transcriptional regulatory element can,for example, comprise one or more promoters, one or more responseelements, one or more negative regulatory elements, and/or one or moreenhancers.

As used herein, a “transcription factor recognition site” and a“transcription factor binding site” refer to a polynucleotidesequence(s) or sequence motif(s) which are identified as being sites forthe sequence-specific interaction of one or more transcription factors,frequently taking the form of direct protein-DNA binding. Typically,transcription factor binding sites can be identified by DNAfootprinting, gel mobility shift assays, and the like, and/or can bepredicted on the basis of known consensus sequence motifs, or by othermethods known to those of skill in the art.

As used herein, the term “operably linked” refers to a linkage of two ormore polynucleotides or two or more nucleic acid sequences in afunctional relationship. A nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. For instance, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

“Transcriptional unit” refers to a polynucleotide sequence thatcomprises at least a first structural gene operably linked to at least afirst cis-acting promoter sequence and optionally linked operably to oneor more other cis-acting nucleic acid sequences necessary for efficienttranscription of the structural gene sequences, and at least a firstdistal regulatory element as may be required for the appropriatetissue-specific and developmental transcription of the structural genesequence operably positioned under the control of the promoter and/orenhancer elements, as well as any additional cis sequences that arenecessary for efficient transcription and translation (e.g.,polyadenylation site(s), mRNA stability controlling sequence(s), etc.

The oligonucleotides (or “ODNs” or “polynucleotides” or “oligos” or“oligomers” or “n-mers”) of the present invention are preferablydeoxyoligonucleotides (i.e. DNAs), or derivatives thereof;ribo-oligonucleotides (ie. RNAs) or derivatives thereof; or peptidenucleic acids (PNAs) or derivatives thereof.

The term “substantially complementary,” when used to define either aminoacid or nucleic acid sequences, means that a particular subjectsequence, for example, an oligonucleotide sequence, is substantiallycomplementary to all or a portion of the selected sequence, and thuswill specifically bind to a portion of an MRNA encoding the selectedsequence. As such, typically the sequences will be highly complementaryto the MRNA “target” sequence, and will have no more than 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 base mismatches throughout the complementary portionof the sequence. In many instances, it may be desirable for thesequences to be exact matches, ie. be completely complementary to thesequence to which the oligonucleotide specifically binds, and thereforehave zero mismatches along the complementary stretch. As such, highlycomplementary sequences will typically bind quite specifically to thetarget sequence region of the mRNA and will therefore be highlyefficient in reducing, and/or even inhibiting the translation of thetarget mRNA sequence into polypeptide product.

Substantially complementary oligonucleotide sequences will be greaterthan about 80 percent complementary (or ‘% exact-match’) to thecorresponding MRNA target sequence to which the oligonucleotidespecifically binds, and will, more preferably be greater than about 85percent complementary to the corresponding mRNA target sequence to whichthe oligonucleotide specifically binds. In certain aspects, as describedabove, it will be desirable to have even more substantiallycomplementary oligonucleotide sequences for use in the practice of theinvention, and in such instances, the oligonucleotide sequences will begreater than about 90 percent complementary to the corresponding mRNAtarget sequence to which the oligonucleotide specifically binds, and mayin certain embodiments be greater than about 95 percent complementary tothe corresponding MRNA target sequence to which the oligonucleotidespecifically binds, and even up to and including 96%, 97%, 98%, 99%, andeven 100% exact match complementary to all or a portion of the targetMRNA to which the designed oligonucleotide specifically binds.

Percent similarity or percent complementary of any of the disclosedsequences may be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0, available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch (1970).Briefly, the GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) that are similar, divided bythe total number of symbols in the shorter of the two sequences. Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and anadditional 0.10 penalty for each symbol in each gap; and (3) no penaltyfor end gaps.

5. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, thcse of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1

Generation of Improved rAAV Vectors

In the present example, rAAV-mediated transduction has been enhanced byusing alternative promoters, such as the human insulin promoter, andrAAV capsid mutants that incorporate a ligand derived fromapolipoprotein E (ApoE) that is targeted to the low density lipoproteinreceptor (LDL-R) (Datta et al., 2000). These studies indicate that thetransduction efficiency can be enhanced several thousand-fold, allowingfor the use of MOIs as low as 5 i.u. per cell. These studies demonstratethe use of modified rAAV vectors for islet cell transduction.

5.1.1 Materials and Methods

5.1.1.1 Plasmid Constructs and rAAV Packaging

The rAAV serotype 2 (rAAV2) vector plasmids used for these studies aredepicted diagrammatically (FIG. 1). Briefly, the CMV-β-actin promoterfrom pCB-hAAT (Xu et al., 2001), the elongation factor promoter and thehuman insulin promoter were cloned into the KpnI and HindIII sites ofpTR-CMV-lucEYFP replacing the CMV promoter.

The rAAV-ApoE construct was made by inserting an oligonucleotide thatcoded for the human Apo E amino acids LRKLRKRLLR (SEQ ID NO:1) andDWLKAFYDKVAEDLDEAF (SEQ ID NO:21), which code for the hApoE LDL-receptorligand and the lipid-associated peptide, respectively, immediately afteramino acid 138 of the VP1 coding sequence. The ApoE-encodingoligonucleotide was flanked by the restriction sites for EagI and MluIand was inserted into pIM45-EM138-ApoE. pIM45 was described previouslyand consists of the AAV nucleotides coding for the rep and cap genes butis missing the terminal repeats (McCarty et al., 1991).

To construct pIM45-EM138, site directed mutagenesis was used to insertan EagI/MluI cloning site immediately after amino acid position 138 ofthe VP1 coding sequence, which is also immediately after the initiatorthreonine codon of VP2. The oligonucleotide sequence was:5′-AGGAACCTGTTAAGACGCGGCCGACGCGTGCTCCGGGAAAAAAGAG-3′ (SEQ ID NO:34) andits complement were used to insert the EagI/MluI restriction site andthe resulting pIM45-EM138 plasmid was sequenced to insure no fortuitousmutations were introduced. The net effect was that pIM45-EM138-ApoEcontains the ApoE receptor ligand and lipid-associated peptide flankedby RP and TR (coded by the EagI and Mlul sites), inserted immediatelyafter the threonine start codon for VP2, which is immediately afteramino acid 138 of VP1.

rAAV production was performed as previously described (Zolotukhin etal., 1999). The method involves cotransfection with two plasmids bycalcium phosphate coprecipitation of a permissive human cell line(HEK293). HEK293 cells were grown as monolayers (initially seeded with6×10⁸ cells per Nunc® cell factory) in Dulbecco's modified minimalessential media (DMEM) containing 10% fetal bovine serum (37° C., 5%CO₂). After 18 h, the cells were transfected with different pairs ofplasmids. The first nonrescuable helper plasmid (pDG) contained therAAV2 complementing functions, rep and cap, as well as the Ad helpergenes (E2a, VA RNA, E4) required for helper function. The second vectorcontained a eukaryotic expression cassette and flanking ITRs.

Transfected cells were maintained at 37° C. in culture (5% CO₂) for 60hr before harvest. Cells were then dissociated by treatment with EDTA,pelleted, resuspended in lysis buffer (20 mmol/l Tris, pH 8.0; 150mmol/l NaCl; 5% deoxycholate) containing benzonase (Merck, Darmstadt,Germany), and incubated for 30 min (37° C., 5% CO₂). Crude lysates wereclarified by centrifugation with virus-containing supernatant purifiedby iodixanol density gradient centrifugation, followed by heparinaffinity chromatography and concentration.

Purity of preparations was determined by subjection of a sample tosilver-stained SDS-PAGE. Infectious center assays were used to determinethe rAAV titer, and dot blot assays were performed to quantify the titerof the rAAV physical particles and then confirmed by real-time PCR. Thelatter values were used to calculate the particle-to-infectivity ratio(Zolotukhin et al., 1999). rAAV-ApoE virus was prepared in essentiallythe same way except that the helper plasmids used were pIM45-EM138-ApoE(to provide a capsid that contained the ApoE ligand inserted into aaposition 138 of VP1) and pXX6 (Xiao et aL, 1996) to provide theadenovirus helper functions.

5.1.1.2 Human and Murine Islet Cell Cultures

Pancreatic islet cells were isolated as previously described (Flotte etal., 2001). Briefly, after intraductal injection of a solutioncontaining Liberase (Boehringer-Mannheim Biochemicals, Indianapolis,Ind.), a whole human pancreas was subjected to mechanical shaking, andaliquots of eluate were withdrawn at various points during a 2-hrperiod. Purification of the final islet preparation was obtained bycentrifugation on discontinuous Eurocollins-Ficoll gradients followed byhand picking. Mouse islets (C57B1/6; Jackson Research Laboratories, BarHarbor, Me.) were obtained through intraductal injection of collagenasetype XI solution (Sigma Chem. Co., St. Louis, Mo.), followed bypurification through repeated washings and hand picking. Islets weremaintained in standard culture conditions (CMRL Medium-1066 with 10%fetal bovine serum; 5% CO₂, 37° C.; D-glucose concentration=3.3 mM).Islet purity was assessed by diphenylthiocarbazone staining, andviability was determined by staining with propidium iodide andfluorescein diacetate.

5.1.1.3 Transduction and Detection of Gene Expression

Intact islets, maintained as previously indicated at concentrations of0.2-1×10³, were transduced at an MOI of 5 to 10,000 infectious units(i.u.) per cell of the appropriate rAAV construct. Islet equivalentswere determined for all pancreatic isolations; an estimate of 2,000cells per islet equivalent was used for all calculations. Specifically,an islet equivalent (i.e., a combination measure of size and number) wasdefined as an islet that was spherical in shape and 150 μm in diameter;an appropriate algorithm was used to calculate the islet equivalentnumber. Using this islet equivalent value, 2×10⁴ to 2×10⁷ i.u. of rAAVhave been used per islet equivalent, which equates to an MOI of 10 to10,000 i.u. per islet cell. Infections of islet cells were performed in16-well chamber slides.

The level of human α-1-antitrypsin (HAAT) was determined byenzyme-linked immunoassay (ELISA). Microtiter plates (Immulon 4®; DynexTechnologies, Chantilly, Va.) were coated with 100 μl of goat anti-hAAT(1:200 diluted; Sigma Immunochemical, St Louis, Mo.) in Voller's bufferovernight at 4° C. Duplicate standard curves (hAAT; SigmaImmunochemical) and serially diluted unknown samples were incubated inthe plate at 37° C. for 1 h. After blocking with 3% bovine serum albumin(BSA), a second antibody, rabbit anti-hAAT (1:1000 diluted, RocheMolecular Biochemicals, Indianapolis, Ind.) was reacted with thecaptured antigen at 37° C. for 1 h. A third antibody, goat anti-rabbitIgG conjugated with peroxidase (1:800 diluted; Roche MolecularBiochemicals) was incubated at 37° C for 1 hr. The plate was washed withPBS-Tween 20™ between reactions. After reaction with the substrate(o-phenylenediamine dihydrochloride, Sigma Immunochemical), plates wereread at 490 nm on an MRX microplate reader (Dynex Technologies).Fluorescent microscopy was performed using a Zeiss Axioskop, andconfocal microscopy was performed using a Bio-Rad 1024ES laser scanningconfocal system attached to an Olympus SX70 inverted microscope.

5.1.1.4 Statistical Analysis

ELISA data from the transduction experiments are represented as mean±SD.ANOVA was used to compare the mean in the different groups andStudent-Newman-Keuls Multiple Comparisons Test was performed. Data areconsidered significant at P<0.05.

5.1.1.5 Portal Injection of rAAV Vectors

All animal procedures were performed with prior approval of theUniversity of Florida Institutional Animal Care and Use Committee. Youngadult, 25-gm C57B16 mice were anesthetized with isoflorane inhalationand underwent laparatomy under aseptic conditions. The portal vein wasdirectly visualized and vector was injected through a 29-gauge stainlesssteel needle. The laparatomy incision was closed and the animals wereallowed to recover. Serial tail vein phlebotomies were performed atbiweekly intervals, and human AAT was measured using a species-specificELISA.

5.1.2 Results

5.1.2.1 Optimization of Transcriptional Activity

Previous studies of rAAV-mediated islet cell transduction have utilizedeither the cytomegalovirus (CMV) immediate early promoter or the CMVenhancer/chicken β-actin hybrid promoter (CB) (Flotte et al., 2001). Inorder to determine whether the transcriptional activity of the rAAVvector cassette could be further enhanced, a series of reporter geneconstructs were prepared, utilizing a translational fusion between thefirefly luciferase gene and the enhanced yellow fluorescent protein(luc-EYFP). The following promoters were evaluated, CMV, CB, elongationfactor 1-α (E1α), and the human insulin promoter (Ins) (FIG. 1). Humanislet cells were transfected using Lipofectamine 2000™. Expression wasmeasured 48 hr later by luminometry. As shown in FIG. 2, the humaninsulin promoter was, by far, the most efficient promoter tested,mediating expression levels at least 10-fold higher than those obtainedwith the CMV or CB promoters. The effect was the same whether or notislets underwent additional treatment with trypsin to enhancepenetration of liposomes.

5.1.2.2 Evaluation of Different rAAV Serotypes

Different serotypes of AAV bind to different cell surface receptors,including heparan sulfate proteoglycan for AAV2 and AAV3 (Summerford andSamulski, 1998), O-linked sialic acid for AAV4 (Kaludov et al., 2001),and N-linked sialic acid for AAV5 (Walters et al., 2001; Auricchio etal., 2001). In an initial comparison of the islet cell transductionefficiency of the various serotypes, a CB-promoter-driven humanα1-antitrypsin (CB-hAAT) cassette was utilized (FIG. 1) as a secretedreporter to transduce murine islets in culture. As shown in FIG. 3, thelevel of HAAT expression achieved 6 days after transduction wassubstantially higher with vector packaged in AAV1 capsids as comparedwith the other serotypes.

The density and composition of cell surface receptors can differsignificantly between species. The above findings were thus confirmedwith human islets, using the green fluorescent protein (GFP) as areporter for AAV1 and AAV2 and the red fluorescent protein (dsRed) forAAV5 (using the appropriate excitation/detection filter set). Confocalmicroscopy revealed no enhancement of gene expression from alternativeAAV serotypes in human islets. This is in contrast with a markedpreference for rAAV1 shown above in murine islets (FIG. 3).

5.1.2.3 Packaging of rAAV2 Genomes into Alternative Capsids

The difference in transduction efficiency between serotypes suggeststhat receptor binding is a limiting step for transduction of islets.Vector preparations are characterized in terms of their physical titerby both DNA dot-blot hybridization and by Taqman® real-time PCR and interms of their biological titer by infectious center assay on C12 cells(an AAV-Rep-expressing Hela cell line). A modest decrease in packagingefficiency was noted with some of these constructs (Table 5). Theinterpretation of infectious center assay data is difficult to interpretin this context, since the abundance of the various receptors on thesecells has not been characterized. However, it is important to note thatthe mutation site is far removed from the heparin-binding domain andshould not create direct steric interference with the native uptakepathway. The infectious center data is included since the particle toinfectious unit ratio can serve as an indicator of partially assembledor unstable vector particles (Wu et al., 2000). TABLE 3 PACKAGING OFRAAV2 VECTOR GENOMES INTO ALTERNATIVE SEROTYPES OR TARGETED MUTANTCAPSIDS Biological Particle to Physical Titer Titer Infectious UnitCapsid Vector Cassette (particles/ml) (i.u./ml) Ratio Wild-type CB-hAAT8.0 × 10¹⁰ 4.3 × 10⁹  19 Wild-type Ins-lucEYFP 3.3 × 10¹² 8.5 × 10¹⁰ 38ApoE CB-hAAT 1.8 × 10¹² 2.3 × 10⁹  782 ApoE Ins-lucEYFP 1.8 × 10¹² 1.4 ×10¹⁰ 126*These values represent purified stocks, while cleared lysates were usedfor the original transduction experiments.5.1.2.4 A Targeting rAAV2 to the LDL-R on Human Islets

In order to evaluate the potential limitation to rAAV vectortransduction that might be mediated by capsid binding to the cellsurface, rAAV2 vector genomes were packaged into a number of alternativecapsids, including AAV serotype 1, 3, 4 and 5 capsids (Rabinowitz etal., 2002) and rAAV2 capsids into which the 28-amino acid ApoE-derivedligand was inserted. Residues within the AAV2 capsid have previouslybeen identified into which new peptides can be inserted, thus allowingone to target specific receptors without disrupting the integrity of thecapsid (Wu et al., 2000). In order to target the LDL-R on islets, aligand derived from ApoE (Datta et al., 2000; Perrey et al., 2001) wasinserted into a site one residue downstream from the N-terminalmethionine of VP2 (FIG. 4). Since VP1 simply represents an N-terminalextension of VP2, this new peptide will be displayed both within VP1 andVP2. Two different reporters were packaged within the rAAV2-ApoEcapsids, a human insulin promoter-driven GFP (Ins-GFP) cassette and thesame CB-hAAT cassette described above. In the GFP transduction studies,the ApoE capsid appeared substantially more efficient for islet celltransduction, with a greater number of cells demonstrating native GFPfluorescence within each islet examined.

The enhancement of transduction was quantified in the hAAT expressionexperiments. Equal volumes of CB-hAAT packaged into either wild-typeAAV2 capsids or AAV2-ApoE capsids were used to infect murine islet cellsand the release of HAAT into the supematant medium was measured at 6 and12 days by ELISA. As shown in FIG. 5A and FIG. 5B, the transductionefficiency was 90-fold greater (945 vs. 11 ng/ml) with the ApoE insert.When the infectious titer of this vector was taken into account,however, the relative transduction efficiency in terms ofexpression/infectious MOI was approximately 9000-fold greater with theApoE capsid. This degree of enhancement is deduced since an equal volumeof a stock with a 100-fold lower infectious titer was used to generate90-fold greater hAAT expression (Table 6). Even if one makes the mostconservative assessment of the enhancement factor, considering thephysical titer rather than the infectious titer, the expression/particlewas enhanced by 220-fold, since the ApoE stock had a physical particletiter 2.3-fold lower than the wt-AAV2 capsid stock. Taken together,these data convincingly demonstrate that receptor targeting can greatlyenhance rAAV transduction, regardless of the promoter or reporter genesystem used. TABLE 6 TRANSDUCTION OF MURINE ISLETS WITH THE RAAV-CB-HAATCASSETTE PACKAGED INTO EITHER WILD-TYPE (AAV2) OR LDL-R TARGETED (APOE)CAPSIDS MOI (based Equivalent on infections MOI (based AAT ExpressionVolume center assay in on physical (ng/ml of Capsid Added C12 cells)titer) medium) Wild-type 10 μl 537.5 10,000 10 Wild-type  1 μl 53.751,000 7 ApoE 10 μl 5.5 4,300 945 ApoE  1 μl 0.55 430 73 ApoE 0.1 μl 0.055 43 25.1.2.5 Targeting rAAV2 to the LDL-R on Human Islets

In order to determine whether LDL-R targeting would enhance genetransfer and expression in vivo, the portal veins of C57B16 mice wereinjected with equal physical particle titer doses of rAAV-CB-hAATpackaged into wild-type AAV2 or ApoE-ligand containing capsids, andserum HAAT levels were examined at timed intervals up to 5 weeks afterinjection. As expected from previous studies, the lower dose of 7.5×10⁹physical particles of the native capsid resulted in no detectable hAATexpression (FIG. 6), while this dose of targeted vector mediatedsignificantly higher levels. At the 10-fold higher dose, the expressionof targeted vector was found to be 4-fold higher than that of thestandard rAAV2 vector. It should be noted that these doses wereapproximately 100-fold lower than those previously reported for optimalin vivo rAAV2-mediated transduction of hepatocytes.

5.1.3 Discussion

Previous work has demonstrated the utility of rAAV for long-termexpression with a minimum of vector-related side effects. Theperformance profile of this vector system in human islets was somewhatmixed, however, since very high MOIs were needed for efficienttransduction (Flotte et al., 2001). In this example, greatly enhancedtransduction efficiency has been demonstrated either by increasingexpression with more active promoters or by increasing cell attachmentand uptake in a very specific manner, targeting the LDL-R with a peptidederived from ApoE. The latter capsid yielded vector preparations withmodestly reduced titer (approximately 10-fold lower than wild-type), butresulted in a dramatic enhancement of transduction efficiency (900-foldas judged by physical titer).

Several groups have reported the use of receptor targeting in thecontext of rAAV in the recent past (Wu et al., 2000; Bartlett et al.,1999; Shi et al., 2001; Nicklin et al., 2001; Grifman et al., 2001).Previously, cell types that have been targeted include hematopoieticprogenitors (Yang et al., 1998), bronchial epithelial cells (Wu et al.,2000), and endothelial cells (Nicklin et al., 2001). The current reportrepresents the greatest increase in transduction efficiency yet observedfrom such a capsid modification, and further demonstrates the in vivoutility of this approach. The sites within AAV where inserts have beensuccessfully placed have generally clustered near the N-terminus (Wu etal., 2000) and within the putative heparin binding domain (especiallypositions 1587 or R588 (Nicklin et al., 2001)). In general, these capsidmutants have a mildly decreased packaging efficiency, as was noted withthe ApoE insert. The relative enhancement by use of AAV1 in mouse isletsis most likely due to targeting of different receptors. However, it isalso possible that the capsid variants could affect other factors suchas the internalization of vector, nuclear targeting or nuclear entry. Itis also worth noting the species-related differences in serotypepreferences. AAV1 capsid was clearly superior to AAV2 in murine islets,while this was not the case in human islets. This illustrates onepotential advantage of targeting a specific receptor known to be in highabundance on the islet cell across species, like the LDL-R. In makingthese comparisons, the most conservative method of comparing physicalparticle titers was chosen. It should be noted, however, that non-nativecapsids could affect particle stability and infectivity in a fashionthat might be reflected in a truly altered particle to infectious unitratio. Therefore, infectious titer information was presented as well.

The use of the human insulin promoter was also found to have asignificant advantage in the overall efficiency of transgene expression.This result has been reported previously by Yang and Kotin (2000). Inthe latter report, the insulin promoter was shown to be glucosesensitive. While the glucose-responsiveness of these constructs were notevaluated, this feature represents a potential mechanism for regulationof the production of therapeutic molecules. The relative specificity ofthe insulin promoter for β cells also adds another level of precision tothe gene delivery process, in that other cells transduced with insulinpromoter-driven constructs are not likely to express the transgene atsignificant levels. It is also very unlikely that the insulin promoterwill undergo transcriptional silencing.

Overall, these studies make it much more practical to consider ex vivoislet cell transduction in the context of islet cell transplantation.Primary candidate genes include IL-10, IL-1 receptor antagonist,antioxidants (such as heme oxygenase and manganese superoxide dismutase[Mn SOD], and inhibitors of apoptosis (Pileggi et al., 2001).

5.2 EXAMPLE 2

Efficient Ex Vivo Transduction of Pancreatic Islet Cells with ImprovedrAAV Vectors

Attempts to use islet cell transplantation for reversing type 1 diabeteshave been documented for more than two decades; however, the procedurehas been largely unsuccessful (Kenyon et al., 1998; Weir andBonner-Weir, 1998). Concurrent mechanisms believed to underlie this lackof success include rejection, recurrence of anti-islet cellautoimmunity, and nonspecific islet loss because of perturbation of thegraft microenvironment (e.g., inflammation, ischemia/reperfusion).

A number of candidate gene products may prevent immune-mediateddestruction and extend graft survival (e.g., interleukin [IL]-4,manganese superoxide dismutase, Bcl-2) (Giannoukakis et al., 1999).Furthermore, these genes may prove safer and more effective thansystemic pharmacological immunosuppression because some agents arethemselves potentially prodiabetogenic (e.g., cyclosporine, FK506,steroids) through imposition of increased metabolic demand. However,such studies have been limited by the lack of gene transfer vectors thatare safe, efficient and long lasting (Fry and Wood, 1999). Recombinantadeno-associated virus (rAAV) vectors have recently demonstrated somesuperiority to other viral and nonviral systems with regard to their invivo safety, efficiency, and duration of action both in animal modelsand in early persistent infections in humans without known pathology andwith only modest immune responses (Carter and Flotte, 1996; Rabinowitzand Samulski, 1998; Bems and Giraud, 1996; Song et al., 1998; Greelishet al., 1999; Hemandez et al., 1999). rAAV retains these beneficialproperties and therefore has the potential to be an ideal vector for invivo gene transfer. However, previous studies have failed to demonstraterAAV transduction of islet cells (Giannoukakis et al., 1999).

5.2.1 Materials and Methods

5.2.1.1 Islet Isolation

Pancreatic islet cells were isolated as previously described (Ricordi etal., 1988). Briefly, after intraductal injection of a solutioncontaining Liberase (Boehringer-Mannheim Biochemicals, Indianapolis,Ind.), a whole human pancreas was subjected to mechanical shaking, andaliquots of eluate were withdrawn at various points during a 2 hrperiod. Purification of the final islet preparation was obtained bycentrifugation on discontinuous Eurocollins-Ficoll gradients followed byhand picking. Mouse islets (C57B1/6; Jackson Research Laboratories, BarHarbor, Me.) were obtained through intraductal injection of collagenasetype XI solution (Sigma, St. Louis, Mo.), followed by purificationthrough repeated washings and hand picking. Islets were maintained instandard culture conditions (human-CMRL-1,066 with 5% normal humanserum; mouse RPMI-1640 with 10% fetal bovine serum; 5% CO₂, 24° C.)until used (within 48 hr). Islet purity was assessed bydiphenylthiocarbazone staining, and viability was determined by stainingwith propidium iodide and fluorescein diacetate.

5.2.1.2 Plasmid Construction, Viral Packaging and Production, andCellular Transduction

The rAAV serotype 2 (rAAV2) vector plasmids used for these experimentsare depicted diagrammatically (FIG. 7A, FIG. 7B and FIG. 7C). Briefly,murine cDNAs for the cytokines IL-4 and IL-10 were cloned into the p43.2(AAV2-ITR-containing-vector) plasmid between the XbaI site downstreamfrom the cytomegalovirus (CMV) promoter and the XbaII site upstream fromthe simian virus 40 (SV40) polyadenylation signal.

rAAV2 production was performed as previously described (Zolotukhin etal., 1999). The method involves cotransfection with two plasmids bycalcium phosphate coprecipitation of a permissive human cell line(HEK293). HEK293 cells were grown as monolayers (initially seeded with6×10⁸ cells) in Dulbecco's phosphate-buffered saline (PBS) containing 5%fetal bovine serum (37° C., 5% CO₂). After 18 hr, the cells weretransfected with different pairs of plasmids. The first nonrescuablehelper plasmid (pDG) contained the rAAV2 complementing functions, repand cap, as well as the Ad helper genes (E2a, VA RNA, and E4) requiredfor helper function. The second vector contained a eukaryotic expressioncassette and flanking inverted terminal repeats (ITRs). Transfectedcells were maintained at 37° C. in culture (5% CO₂) for 60 hr beforeharvest. Cells were then dissociated by treatment with EDTA, pelleted,resuspended in lysis buffer (20 mmol/l Tris, pH 8.0; 150 mmol/l NaCl; 5%deoxycholate) containing benzonase (Merck), and incubated for 30 min(37° C., 5% CO₂). Crude lysates were clarified by centrifugation withvirus-containing supernatant purified by iodixanol density gradientcentrifugation, followed by heparin affinity chromatography andconcentration. The purity of preparations was determined by subjectingthe sample to silver-stained SDS-PAGE. Infectious center assays wereused to determine the rAAV titer, and dot blot assays were performed toquantify the titer of the rAAV physical particles andparticle-to-infectivity ratio (Zolotukhin et al., 1999). Intact islets,maintained as previously indicated at concentrations of from about0.2×10³ to about 1×10³, were transduced at a multiplicity of infection(MOI) of 10 to 10,000 infectious units (i.u.) per cell of theappropriate rAAV construct. Islet equivalents were determined for allpancreatic isolations; an estimate of 2,000 cells per islet equivalentwas used in all calculations. Specifically, an islet equivalent (i.e., acombination measure of size and number) was defined as an islet that wasspherical in shape and 150 μm in diameter, an appropriate algorithm wasused to calculate the islet equivalent number. Using this isletequivalent value, from about 2×10⁴ to about 2×10⁷ i.u. of rAAV were usedper islet equivalent, which equated to an MOI of 10-10,000 i.u. perislet cell. For studies using adenovirus (Ad) as a helper virus, isletcells were treated with adenovirus 5 (Ads) at an MOI of 5 for 2 hr (37°C., 5% CO₂) before confection with rAAV.

The comparison of rAAV2 and rAAV serotype 5 (rAAV5) vectors wasperformed using an expression cassette consisting of a Rous sarcomavirus (RSV) long-terminal repeat promoter and a nuclear-targetedβ-galactosidase (nlacZ) transgene, flanked by either rAAV2-ITRs (Afioneet al., 1999) or rAAV5-ITRs (Chiorini et al., 1999). The rAAV2-nlacZconstruct was packaged as described above, by cotransfection of thevector plasmid with the 5RepCapB helper plasmid (Chiorini et al., 1999)into AdS-infected cos cells and purified by CsCl ultracentrifugation.

5.2.1.3 Measurement of Cytokine and Insulin Production

Microtiter plates (Immulon 4®) were coated with 50 μl of a 1:250dilution of anti-mouse IL-4 or IL-10 (#265113E, #26571E; Pharmingen, SanDiego, Calif.) in 0.1 mol/l sodium bicarbonate buffer (overnight, 4°C.). After washing and appropriate blocking (with 10% fetal bovine serumin PBS-Tween 20™ , 1 hr at 24° C.), standards for IL-4 or IL-10 andtissue culture medium samples were incubated in the plate at 24° C. for1 hr. After washing, a second antibody (1:250 dilution of horseradishperoxidase-conjugated anti-mouse IL-4, #26517E, or 1:250 dilution ofbiotylated anti-mouse IL-10, #26572E, with streptavidin-horseradishperoxidase conjugate) was reacted with the captured antigen at 24° C.for 1 hr. After extensive washing, detection was performed using a thirdincubation with the absorbance at 490 nm. For the detection of insulin,supernatants were processed and hormone secretion was quantitated usingcommercial kits (Mercodia, Minneapolis, Minn.). Data are expressed asmeans±SE.

5.2.1.4 Immunochemistry

For insulin immunolocalization, intact human islets were ethanol fixedand rehydrated through repeated washings in solutions containingdecreasing ethanol concentrations (99, 95, 70, and 0%; 30 s, 24° C.).After being washed in PBS, islets were incubated for 1 hr at 24° C. with0.5 μg/ml guinea pig monoclonal anti-insulin antibody (Dako). Primaryantibody was detected after standard washing and blocking steps,including incubation (1 hr, 24° C.) with biotinylated goat anti-guineapig antibody coupled thereafter with streptavidin-RPE-CyS (Daka.).Fluorescent microscopy was performed using a Zeiss Axioplat unit, andconfocal microscopy was performed using a Bio-Rad 1024ES laser scanningconfocal system attached to an Olympus SX70 inverted microscope.

5.2.2 Results and Discussion

rAAV binds to cells via a heparan sulfate proteoglycan receptor. Afterit has been attached, its entry is dependent on the presence of acoreceptor, which may consist of either the fibroblast growth factorreceptor or the α_(v)-β₅ integrin molecule (Summerford and Samulski,1998; Summerford et al., 1999). To readdress the question of whetherislet cells were permissive for rAAV vectors, a series of transductionstudies with purified human islets was performed. These initial studiesused both the UF5 rAAV-CMV-green fluorescent protein (GFP) vector andthe UF11 rAAV-CMV/β-actin (CB) hybrid promoter-GFP vector (Zolotukhin etal., 1999). Batches of 1×10³ intact human islets were infected at an MOIof 10 to 10,000 i.u. per islet equivalent. To enhance scientificinterpretation of these short-term in vitro studies, islets werecoinfected with Ad5 at an MOI of 5. This coinfection procedure resultsin an acceleration of leading strand synthesis (Bems and Giraud, 1996;Afione et al., 1999) but is not an absolute requirement for rAAVtransgene production. Standard fluorescent as well as confocalmicroscopy revealed that GFP expression was quite efficient (i.e., >40%GFP-positive cells by computer-aided morphologic assessment) in humanislets within 48 hr of infection under these conditions Interestingly,transduction was much less efficient (<1% GFP-positive cells) at an MOIof 1,000, was indistinguishable from control vector at an MOI of 100 orless, and was of similar efficacy (at equivalent MOI) using either CMV-or CB promoter-based systems. It is believed the use of a high MOI,combined with benefits afforded through recent improvements in rAAVpurification methods (Zolotukhin et al., 1999), led to this novelfinding of islet cell rAAV transduction, which was not observed inprevious studies (Giannoukakis et al., 1999).

Although the successful transduction of human islets represents animportant finding in terms of affording the feasibility for futureclinical intervention in humans, identifying rAAV transduction in rodentislets is also considered crucial for investigations exploring theeffects of therapeutic transgene expression in experimentaltransplantation models. To address the issue of species specificity andpermissiveness for islet rAAV transduction, identical studies (both interms of MOI titration and testing of CMV and CB promoters) wereextended to intact islet cells obtained from mice. Similar to humanislets, successful transduction (as demonstrated by rAAV-GFP expressionwas achieved with titration and promoter efficiencies overlapping thoseobserved with their human cellular counterparts.

Another key issue for therapeutic efficacy concerns the distribution ofrAAV-GFP expression within an islet in combination with the question ofwhether rAAV transduction of β cells occurs. To address these issues, aseries of intact islets were transduced with rAAV-GFP and subjected toconfocal imaging (single slice of a transduced human islet, a procedurethat revealed homogenous GFP expression throughout the islet. Toidentify whether β cells were capable of transduction, cytocentrifugedpreparations of these rAAV-GFP-transduced human islet cells wereimmunostained with an RPE-Cy5-conjugated anti-insulin antibody.Fluorescence microscopy revealed colocalization of staining in β cells(red anti-insulin stain, green rAAV-GFP fluorescence), indicating thatthis cell type had been effectively transduced.

Depending on the mode of administration (i.e., systemic versus local),treatment with the immunoregulatory cytokines IL-4 and IL-10 can inhibitthe recurrence of type 1 diabetes (alloimmune and/or autoimmune) in micereceiving islet transplants; IL-4 seems to inhibit disease-causinglymphocytes and IL-10 seems to limit the activation of potentialdiabetogenic CDS⁺ T-cells (Rabinovitch et al., 1995; Benhamou et al.,1996; Gallichan et al., 1998). However, use of cytokines for initiationof immune deviation systemically would currently be limited because ofthe need for repeated administration, because of their relatively shorthalf life, and local production, which is depending on the availabilityof suitable targeted gene delivery systems (Schmidt-Wolf andSchmidt-Wolf, 1995; Robbins and Evans, 1996). A modification of isletcells toward production of these anti-inflammatory cytokines, achievableby rAAV gene transfer, is significant in developing novelimmunointervention protocols for type 1 diabetes.

To address this strategy, human pancreatic islets were transduced withrAAV vectors containing the cytokines IL-4 and IL10. Specifically,experiments were performed wherein at an MOI of 10,000, intact humanislets were transduced with rAAV-CMV-IL-4 or rAAV-CMV-IL-10 (FIG. 7A).At 48 hr, both IL-4 and IL-10 were readily detectable from treatedislets (1.32±0.62 and 2.23±0.34 ng/ml per 0.2×10³ islets for IL4 andIL-10, respectively; FIG. 7B), whereas levels of these cytokines werenot detectable from Ad-infected islets transduced with GFP or irrelevantrAAV control vector preparations. These data demonstrated successfulrAAV-mediated islet cell transduction with a potentially therapeuticsecreted protein. It was also of interest to observe whether thetransduction of islets with rAAV interfered with β-cell metabolicfunction. To address this issue, sets of 0.4×10² intact human islets (intriplicate) were used as a control or transduced with the UF5rAAV-CMV-IL-10 vector (MOI 10,000 i.u.) plus Ad5 (MOI 5 i.u.), UF5rAAV-CMV-IL-10 vector (MOI 10,000 i.u.) alone, or Ad5 (MOI 5 i.u.)alone. The islets were maintained for 48 hr under basal (5 mmol/lglucose) or stimulated (20 mmol/l glucose) conditions; medium sampleswere withdrawn at 0, 2, 12, 24, and 48 hr for analysis of insulinproduction. Although conditions of elevated glucose imparted a two- tothreefold increase in insulin release, no differences (analysis ofvariance, NS) in insulin release were detected between the control andthe groups of transduced islets within the two conditions of glucosestimulation (FIG. 7C). Repeat experiments performed with islets fromwhich samples were collected at 24-hr intervals over 1 one-week intervalprovided similar results, in that no differences in insulin release wereidentified. In general, neither wild-type AAV nor rAAV has ever beenshown to induce apoptosis. However, this possibility was formallyexcluded by measuring free lactate dehydrogenase in the supernatantmedia after infection. The lack of a lactate dehydrogenase increaseindicated that islets remained viable throughout the course of theexperiment. In addition, a fine metabolic assessment of islet cellfunction using a static glucose stimulation assay has been performed;studies further support the contention that rAAV-transduced islets arenot impaired in terms of their glucose responsiveness. Specifically,islets respond to low to high-to-low (i.e., 1.67 to 16.7 to 1.67 mmol/l)sequential incubations with appropriate insulin secretion levels.

Although these data were very promising, the very high MOI of rAAVrequired could present a significant limitation to clinical genetherapy. It was hypothesized that a scarcity of receptors for AAV2 onislet cells could account for this. To test this hypothesis, thetransduction efficiency of rAAV2 at a lower MOI (100 i.u.) was comparedwith that of rAAV serotype 5, which uses a different attachment receptor(Chiorini et al., 1999; Davidson et al., 2000; Zabner et al., 2000).Cultures containing 1×10⁵ Ad-infected murine islet cells were transducedwith 1×10⁷ infectious units of either rAAV2-RSV promoter-drivennuclear-localized lacZ vector or 1×10⁷ infectious units ofrAAV5-RSV-nlacZ. These intact cells were cultured for 48 hr and thenstained with X-Gal for 4 hr before imaging. As previously shown, rAAV2was ineffective at this dose, whereas rAAV5 resulted in abundantlacZ-positive nuclei. This finding was consistent with the hypothesisthat AAV2 receptors are limiting and indicated a possible role forrAAV5-based vectors in future studies.

In a preferred embodiment, rAAV islet cell gene therapy is effectiveeven in the absence of Ad augmentation and is stable aftertransplantation. It was hypothesized, based on earlier studies, thatconversion of rAAV from ss-DNA to ds-DNA form in the absence of Ad wouldrequire at least 7-10 days (Afione et al., 1999). Furthermore, studieswith a related rAAV2-α1-antitrypsin vector showed that rAAV expressionin human islets transduced without Ad was measurable at 3.5-fold abovebackground by days 7-8. To more formally test this, a bolus of 1,000pancreatic islets transduced with the UF11 vector in the absence of Adwere transplanted under the renal capsule of syngeneic C57B1/six mice.Mice were sacrificed two weeks later, and the site of the graft wasanalyzed by epifluorescence. Transduced islets showed bright greennative GFP fluorescence, whereas the surrounding kidney parenchyma andcontrol kidney showed very little background autofluroescence. Theefficiency of GFP expression at 2 weeks without Ad was greater than thatseen with Ad at the earlier time points. As with previous in vivostudies with rAAV (Song et al., 1998; Hernandez et al., 1999; Conrad etal., 1996; Flotte et al., 1993), a carefully controlledhistopathological examination of parallel hematoxylin-eosin-stainedsections showed no evidence of inflammation or cellular infiltrationwithin or near the implanted islets.

Despite advances in disease management, life span is shortened byone-third in patients in whom type 1 diabetes develops before age 30years, and many patients develop significant microvascular andmacrovascular complications (Atkinson and Maclaren, 1995). Thesecomplications are the reason diabetes is recognized as a leading causeof blindness (retinopathy), heart disease, peripheral vascular disease,renal failure, and impotence. Because there are no known innovationsthat seem likely to alter this situation soon, a novel approach topreventing pancreatic β-cell destruction after islet celltransplantation is appealing.

The findings of this study demonstrate feasibility for a family ofimproved rAAV vectors that may dramatically improve islet celltransplantation through genetic engineering of islets. The presentmethodology was to devise an efficacious gene delivery system that wouldallow for testing the question of whether immunoprotection could beafforded through local cellular production of immunomodulatorycytokines. This approach is very topical because the role ofimmunomodulatory cytokines, in terms of allograft and xenograftsrejection, and recurrent autoimmunity are subjects of current interestand controversy (Holzknecht and Platt, 2000). However, it should benoted that the introduction of cytoprotective molecules into isletsusing rAAV vectors will not be limited to cytokines. A series of recentstudies has indicated pivotal roles for both antioxidants (e.g.,heme-oxygenase-1, Mn SOD) and agents capable of interrupting apoptoticpathways (e.g., Bcl-2, surviving) in prolonging graft survival aftertransplantation.

5.3 EXAMPLE 3

Mutational Analysis of the AAV2 Capsid Gene and Construction of AAV2Vectors with Altered Tropism

Adeno-associated virus type 2 (AAV2) belongs to the human parvovirusfamily, which requires a helper virus for production replication (Bernsand Bohenzky, 1987; Buller et al., 1981; Casto et al., 1967). Thenonenveloped capsid adopts an icosahedral structure with a diameter ofapproximately 20 nm. Packaged within the capsid is a single-stranded DNAgenome of 4.7 kb that contains two large open reading frames (ORFs), repand cap (Srivastava et al., 1983). Three structural proteins, designatedVP1, VP2, and VP3, are encoded in the cap ORF and made from the p40promoter by use of alternative splicing and alternative start codons.The three proteins share the same ORF and end at the same stop codon.The C-terminal regions common to all three capsid proteins fold into aβ-barrel structure that is present in several viruses (Rossmann, 1989).Their molecular masses are 87, 73, and 62 kDa, and their relativeabundances within the capsid are approximately 5, 5, and 90%,respectively (Muzyczka, 1992). Recently, AAV has attracted a significantamount of interest as a vector for gene therapy (Berns and Giraud, 1995;Muzyczka, 1992). It has a number of unique advantages that arepotentially useful for gene therapy applications, including the abilityto infect nondividing cells, a lack of pathogenicity, and the ability toestablish long-term gene expression.

Early genetic studies on deletion mutants of AAV revealed that capsidproteins were required for accumulation of single-stranded DNA andproduction of infectious particles (Hermonat et al., 1984; Tratschin etal., 1984). Mutations in the C-terminal region common to all threeproteins also abolished virion formation and failed to accumulatesingle-stranded DNA (Ruffing et al., 1994). VP1 was thought to beimportant for virus infectivity or stability because mutations in theN-terminal region unique to VP1 produced DNA-containing particles withsignificantly reduced infectivity (Hermonat et al., 1984; Tratschin etal., 1984). In vitro assembly studies (Ruffing et al., 1992) and capsidinitiation codon mutagenesis studies (Muralidhar et al., 1994) suggestedthat both VP2 and VP3 were required for capsid formation and productionof infectious particles, and either VP1 or VP2 was required for nuclearlocalization of VP3. Recently, Hoque et al. (1999) have shown that theVP2 N-terminal residues 29 to 34 are sufficient for nucleartranslocation and suggested that the major function of VP2 is totranslocate VP3 into the nucleus. A recent insertional mutation study onAAV capsid protein revealed that mutations in the capsid gene couldaffect AAV capsid assembly and infection (Rabinowitz et al., 1999).Since the crystal structure of AAV was still unavailable, the functionaldomains of the AAV capsid proteins were mostly predicted based oninformation derived from other related autonomous parvoviruses, canineparvovirus (CPV), feline panleukopenia virus, and B19, whose crystalstructures were available (Agbandje et al., 1994; Agbandje et al., 1998;Tsao et al., 1991; Tsao et al., 1992). Sequence comparison of AAV tothese viruses revealed a few conserved functional domains (Chapman andRossman, 1993; Chiorini et al., 1997), but the exact functions of thesedomains were not clear.

While certain groups of cells cannot be transduced by AAV (Klein et al.,1998; Ponnazhagan et al., 1997), AAV can transduce a wide variety oftissues, including brain, muscle, liver, lung, vascular endothelial, andhematopoietic cells (Fisher et al., 1996; Fisher-Adams et al., 1996;Flotte et al., 1993; Gnatenko et al., 1997; Kaplitt et al., 1994; Xiaoet al., 1996; Zhou et al., 1994). Recently, Summerford and Samulski(1998) reported that heparan sulfate proteoglycan is the primarycellular receptor for AAV, and their group further revealed that thebinding site lies within VP3 (Rabinowitz et al., 1999). In addition,human fibroblast growth factor receptor 1 and α_(v)β₅ integrin wereidentified as coreceptors for AAV (Qing et al., 1999; Summerford et al.,1999). Attempts to alter the AAV capsid also have been made in order toexpand the tropism of AAV. Yang et al. (1998) showed improvedinfectivity of hematopoietic progenitor cells by generating a chimericrecombinant AAV (rAAV) having the single-chain antibody against humanCD34 protein. Girod et al. (1999) showed that insertion of the L14epitope into the capsid coding region can expand the tropism of thisvirus to cells nonpermissive for AAV infection that bear the L14receptor. However, in both cases, the normal AAV tropism was notdisrupted. Ideally, for the purpose of retargeting, the normal AAVreceptor binding would need to be modified so that rAAV infects onlytargets bearing the receptors for the engineered epitope.

In this example, site-directed mutagenesis was used to mutate the capsidORF. Initially, 48 alanine scanning mutations were made in which two tofive charged amino acids in the AAV capsid ORF were mutated to alanineresidues by site-directed mutagenesis. It was reasoned that since themutations were an average of 15 to 20 amino acids (aa) apart and spannedthe whole capsid gene, some of them would inevitably fall in or near thefunctional domains of AAV capsid. In addition, a library of substitutionand insertion mutations have been made in a search for regions thatcould tolerate insertions for the purpose of retargeting AAV vectors. Byanalyzing these mutants, a preliminary functional map of the AAV capsidprotein was obtained. The results identified critical regions within thecapsid that were potentially responsible for receptor binding, DNApackaging, capsid formation, and infectivity. In addition, sites thatwere suitable for epitope insertions that might be useful for targetedgene delivery were identified.

5.3.1 Materials and Methods

5.3.1.1 Cell Culture

Low-passage-number (passages 27 to 38) HFK 293 cells (Graham et al.,1977) and HeLa cells were grown in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal calf serum, penicillin (100 U/ml), andstreptomycin (100 U/ml) at 37° C. and 5% CO₂. IB3 cells were propagatedas described elsewhere (Song et al., 1998).

5.3.1.2 Construction of AAV Capsid Mutant Plasmids

pIM45 (previously called pIM29-45 [McCarty et al., 1991]) was used asthe template for all mutant constructions. Mutagenesis was achieved byusing the Stratagene site-directed mutagenesis kit according to thesupplier's manual. For each mutant, two PCR™ primers were designed whichcontained the sequence of alanine substitution or insertion plus aunique endonuclease restriction site flanked by 15 to 20 homologous bpon each side of the substitution or insertion. The restriction site wasdesigned to facilitate subsequent DNA sequencing of the mutants and forpotential insertion of tags or foreign epitopes. The PCR™ products weredigested with endonuclease DpnI to eliminate the parental plasmidtemplate and were propagated in Escherichia coli XL-Blue™ (Stratagene).Miniprep DNAs were extracted from ampicillin-resistant colonies and werescreened by restriction endonuclease digestion. Positive clones weresequenced in the capsid ORF region. The capsid ORF was then subclonedback into the pIM45 backbone with SmaI and SphI to eliminate backgroundmutations. The same mutagenesis strategy was used for peptidesubstitution and insertion mutant constructions.

5.3.1.3 Production of rAAV Particles

To produce rAAV with mutant capsid proteins, 293 cells were transfectedwith three plasmids: (i) pIM45, which supplied either wild-type (wt) ormutant capsid proteins (McCarty et al., 1991); (ii) pXX6, whichcontained the adenovirus (Ad) helper genes (Xiao et al., 1998); and(iii) pTRUF5, which contains the green fluorescent protein (gfp) genedriven by the cytomegalovirus (CMV) promoter and flanked by the AAVterminal repeats (Klein et al., 1998). In some experiments, pTRUF5 wassubstituted with CBA-AT, a recombinant AAV plasmid that contains thehuman α1-antitrypsin (hAAT) gene under the control of the CMV-β-actinpromoter. The plasmids were mixed at a 1:1:1 molar ratio. Plasmid DNAsused for transfection were purified using a Maxi-Prep™ kit (Quagen,Inc., Chatsworth, Calif.) according to the manufacturer's instructions.

The transfections were carried out as follows: 293 cells were split 1:2the day before the transfection so that they could reach 75% confluencythe next day. Ten 15-cm diameter plates were transfects at 37° C., usingcalcium phosphate as described elsewhere (Zolotukhin et al., 1999), andincubated at 37° C. Forty-eight hr after transfection, cells wereharvested by centrifugation at 1,140×g for 10 min, the pellets wereresuspended in 10 ml of lysis buffer (0.15 M NaCl, 50 mM Tris-HCl, pH8.5), and viruses were released by freezing and thawing three times. Thecrude rAAV lysates were treated with Benzonase (pure grade; Merck) at afinal concentration of 50 U/ml at 37° C. for 30 min. The crude lysateswere clarified by centrifugation at 3,700×g for 20 min, and thesupernatant was subjected to further purification by iodixanol stepgradient and heparan sulfate affinity purification as previouslydescribed (Zolotukhin et al., 1999).

To determine whether any of the mutants were temperature sensitive, thetransfections were done in six-well dishes as duplicates at 39.5° C. and32° C. Viruses were resuspended in 250 μl of lysis buffer. All cruderAAV preparations were stored at −80° C. until their titers weredetermined.

5.3.1.4 Gel Electrophoresis, Immunoblotting and Immunoprecipitation

Crude or purified rAAV samples were analyzed on sodium dodecyl sulfate(SDS)-10% polyacrylamide gels. The samples were mixed with sample bufferand boiled at 100° C. for 5 min before loading. For immunoblotting, theproteins were transferred to a Nitro-bond membrane at 4° C., and themembrane was probed with monoclonal antibody (MAb) B1, directed againstthe capsid proteins (Wistuba et al., 1997). The capsid bands werevisualized by peroxidase-coupled secondary antibodies using ECL®(enhanced chemiluminescence detection) (Amersham Biosciences,Piscataway, N.J.) as suggested by the supplier.

For immunoprecipitation, heparan column-purified rAAV samples werediluted in 10 volumes of NETN buffer (0.1 M NaCl, 1 mM EDTA, 20 mMTris-HCI (pH 7.5), 0.5% Nonidet P-40®) and incubated overnight at 4° C.in the presence of a MAb to the hemagglutinin (HA) epitope conjugated toSepharose beads (BabCo, CRP, Denver, Pa.). For a negative control, MAbAU1-conjugated beads (BabCo) were used. AU1 is a commonly used epitope,DTYRYI (SEQ ID NO:35). After incubation, the samples were centrifugedfor 5 min at 17,600×g at 4° C. The beads were washed three times with 1ml of NETN for 10 min at room temperature and resuspended in proteinloading buffer. After centrifugation, the supernatant was precipitatedwith 15% trichloroacetic acid on ice for 1 hr and centrifuged for 45 minat 4° C., and the pellet was resuspended in loading buffer. The samplesthen were boiled in sample buffer and analyzed by Western blotting withMAb B1 as described above.

5.3.1.5 Virus Titers

The infectious titers of rAAV-containing wt and mutant capsids weremeasured at two temperatures, 39.5° C. and 32° C., for the alaninescanning mutants and at 37° C. for all other mutants by using thefluorescent cell assay, which detects expression of the gfp gene. Thiswas done essentially as described previously by Zolotukhin et al.(1999). Briefly, 293 cells were seeded in a 96well dish the day beforeinfection so that they would reach about 75% confluence the next day.Serial dilutions of wt and mutant rAAV-GFP crude preparations were addedto the cells in the presence of Ad5 at a multiplicity of infection (MOI)of 10. The cells and viruses were incubated at 37° C. (or 32° C. and39.5° C.) for 48 hr, and the titers were determined by counting thenumber of green cells with the fluorescence microscope. For each mutant,the infections were done twice and the average was taken. For mutantsthat contained a packaged CBA-AT gene, infectivity was measured by theinfection center assay on 293 cells as previously described (Zolotukhinet al., 1999) and by enzyme-linked immunosorbent assay (ELISA)measurement of HAAT secreted into culture media from infected cells asdescribed elsewhere (Song et al., 1998).

To determine the rAAV physical particle titer, the A20 ELISA kit(American Research Products (Belmont, Mass.) was used. The crude rAAVstocks were serially diluted and incubated with the A20 kit plate. Thereadings that fell into the linear range were taken, and the titers wereread off the standard according to the manufacturer's instructions. TheA20 antibody detects both full and empty particles (Wistuba et al.,1995).

To determine the titer of rAAV physical particles that were full (i.e.,contained DNA), the quantitative competitive PCR™ (QC-PCR™) assay wasused as described previously (Zolotukhin et al., 1999). The crude rAAVstocks (100 μl) were digested first with DNase 1 to eliminatecontaminating unpackaged DNA in 50 mM Tris-HCl (pH 7.5)—10 mM MgCl₂ for1 hr at 37° C. and then incubated with proteinase K (Boehringer) in 10mM Tris HCl (pH 8.0)—10 mM EDTA—1% SDS for 1 hr at 37° C. Viral DNA wasextracted twice in phenol-chloroform and once with chloroform and thenprecipitated by ethanol in the presence of glycogen (10%). The DNA waswashed with ethanol, dried, and dissolved in 100 μl of H₂O, and 1 μl ofthe viral DNA was used for QC-PCR™. Serial dilutions of the internalstandard plasmid DNA with a deletion of GFP were included in thereaction, and the PCR™ products were separated by 2% agarose gelelectrophoresis. The densities of the target and competitor bands ineach lane were measured using ZERO-Dscan image analysis system software(Version 1.0; Scanalytics, Fairfax, Va.) to determine the DNAconcentration of the virus stock.

5.3.1.6 Heparan Column Binding Assay

The ability of mutants to bind to heparan sulfate was tested essentiallyas previously described (Zolotukhin et al., 1999). Crude rAAVpreparations containing wt or mutant capsids were first subjected toiodixanol gradient purification. The 40% layer was then collected andloaded onto a 1-ml pre-equilibrated heparan column at room temperature(immobilized on cross-linked 4% beaded agarose; Sigma H-6508). Theflowthrough fraction, wash (3 column volumes), and 1 M NaCl eluate werecollected, and equivalent amounts of each sample were mixed with SDSsample buffer and electrophoresed on SDS-polyacrylamide gels. The yieldof capsid proteins in each fraction was monitored with MAb B1 by Westernblotting and ECL detection.

5.3.1.7 Electron Microscopy

Electron microscopy (EM) was done in the ICBR EM lab of the Universityof Florida. Iodixanol gradient and heparan column-purified wt or mutantGFP-rAAVs were desalted and concentrated by using a Centricon 10 filter(Amicon). About a 5-μl drop of the virus sample was spotted ontocarbon-coated grids and left for 1 min at room temperature. Excess fluidwas drawn off, and the sample was washed three times withphosphate-buffered saline; 5 μl of 1% uranyl acetate was added for 10sec, and the grid was dried at room temperature for 10 min beforeviewing under EM.

5.3.2 Results

5.3.2.1 Generation of AAV Capsid Mutations

The studies were begun by using alanine scanning site-directedmutagenesis in the hope that some of the mutants would be temperaturesensitive (Cunningham and Wells, 1989). The mutants were constructed inthe noninfectious AAV plasmid, pIM45, which contains all of the AAV DNAsequence except the AAV terminal repeats. There are approximately 60charged clusters in the AAV capsid gene. Some of the clusters areoverlapping; in those cases, only one cluster was chosen. For theinitial round of mutagenesis, 48 sites, named mut1 to mut48, weretargeted. These were spaced approximately equally over the capsid gene,with 12 mutants exclusively in VP1, 5 in VP2, and the rest in VP3 (FIG.8). With the exceptions noted below, in each cluster, all charged aminoacids were converted to alanine. The mutations were created so that theyalso contained a restriction site at the site of mutation to facilitateconfirmation of the mutant sequence and subsequent insertion of foreignepitopes (Table 7). In addition, after sequence comparison of AAVserotypes 1 to 6, several other positions were targeted. mut28 and mut35were made at positions where extra amino acids were found in AAV4 bysequence comparison with AAV2. mut32 was made by replacing TTT with AAAsince TTT was not conserved among other AAV serotypes at aa 454.Finally, in mut29 and mut31, only one Arg residue was changed to Ala,and in mut45 and mut48, only one Lys was changed to Ala. The positionsof the alanine scanning mutants and the specific amino acidsubstitutions are summarized in Table 7 and FIG. 8. TABLE 7 SUMMARY OFMUTANTS Mutant^(a) Type^(b) Amino Acid Positions^(b) Class Phenotype^(c)mut1¹ Ala sub 9-13 DWLED-AWLAA 1 wt mut2¹ Ala sub 24-28 KLKPG-ALAPG 1 wtmut3² Ala sub 33-37 KPKER-APAAA 1 wt, surface mut4² Ala sub 39-43KDDSR-AAASA 2a pd, hep⁺ mut5³ Ala sub 63-67 EPVNE-APVNA 2a pd, hep⁺mut6² Ala sub 67-71 EADAA-AAAAA 2a pd, hep⁺ mut7² Ala sub 74-78EHDKA-AHAAA 2a pd, hep⁺ mut8² Ala sub 76-80 DKAYD-AAAYA 2a pd, hep⁺mut9¹ Ala sub 84-88 DSGDN-ASGAN 1 wt mut10² Ala sub 95-99 HADAE-AAAAA 2apd, hep⁺ mut11² Ala sub 102-107 ERLKED-AALAAAA 1 wt mut12² Ala sub122-126 KKRVL-AAAVL 2a pd, hep⁺ mut13² Ala sub 142-146 KKRPV-AAAPV 1 wtmut14¹ Ala sub 152-156 EPDSS-APASS 1 wt mut15² Ala sub 168-172RKRLN-AAALN 2a pd, hep⁺ mut16² Ala sub 178-182 GDADS-GAAAS 1 wt mut17¹Ala sub 180-184 DSVPD-ASVPA 1 wt mut18² Ala sub 216-220 EGADG-AGAAG 2aPd, hep⁺ mut19¹ Ala sub 228-232 WHCDS-WACAS 4b ni, no capsid mut20² Alasub 235-239 MGDRV-MGAAV 4b ni, no capsid mut21⁴ Ala sub 254-258NHLYK-NALYA 2b pd, unstable capsid mut22⁴ Ala sub 268-272 NDNHY-NANAY 4ani, full particle mut23⁴ Ala sub 285-289 NRFHC-NAFAC 4b ni, no capsidmut24² Ala sub 291-295 FSPRD-FSPAA 4b ni, no capsid mut25² Ala sub307-311 RPKRL-APAAL 4b ni, no capsid mut26² Ala sub 320-324 VKEVT-VAAVT3a hs mut27¹ Ala sub 344-348 TDSEY-TASAY 3a hs mut28² Ala sub 384-385AAA 3a cs mut29¹ Ala sub 389 R-A 1 wt mut30² Ala sub 415-419 FEDVP-FAAVP2a pd, hep⁺ mut31⁴ Ala sub 432 R-A 4c ni, empty particle mut32² Ala sub454-456 TTT-AAA 1 wt mut33² Ala sub 469-472 DIRD-AIAA 3a hs mut34² Alasub 490-494 KTSAD-ATSAA 2a pd, hep⁺ mut35² Ala ins 509 AAAA 3b cs, hep⁻,surface mut36¹ Ala sub 513-517 RDSLV-AASLV 2a pd, hep⁺ mut37² Ala sub527-532 KDDEEK-AAAAA 4a ni, full particle mut38² Ala sub 547-551SEKTN-SAATN 1 wt mut39² Ala sub 553-557 DIEKV-AIAAV 2b pd, unstablecapsid mut40² Ala sub 561-565 DEEEI-AAAAI 4d ni, hep⁻, full particle,surface mut41² Ala sub 585-588 RGNR-AGAA 2c pd, hep⁻, surface mut42² Alasub 607-611 QDRDV-QAAAV 4b ni, no capsid mut43² Ala sub 624-628TDGHR-TAGAF 1 wt mut44¹ Ala sub 637-641 FGLKH-FGLAA 1 wt mut45² Ala sub665 K-A 1 wt mut46² Ala sub 681-683 EIE-AAA 4b ni, no capsid mut47² Alasub 689-693 ENSKR-ASSAA 4b ni, no capsid mut48¹ Ala sub 706 K-A 2a Pd,hep⁺ L1 HA ins 266 2a pd, A20⁻, A20 epitope⁻, surface L2 HA ins 328 4ani, A20⁺, surface L3 HA ins 447 2a pd, hep⁺, surface L4 HA ins 522 4dni, hep⁻, surface L5 HA ins 553 4a ni, A20⁺, surface L6 HA ins 591 2cpd, hep⁻, surface L7 HA ins 664 2a pd, hep⁺, surface VPN1 HA, AU ins  12a pd, hep⁺, surface VP1 HA ins, Ser  34 2a pd, hep⁺, surface subVPN2^(d) HA, Ser ins 138 2a pd, hep⁺, surface VPN3 HA, Ser ins 203 4bni, no capsid VPC HA, Ser, 735 4b ni, no capsid AU, His ins mut1subser1Ser sub  10 4a ni, A20⁺ mut2subser2 Ser sub  24 4a ni, A20⁺ mut3subser3Ser sub  34 2a pd, hep⁺ mut9subser4 Ser sub  84 4a ni, A20⁺ mut14subser5Ser sub 150 4a ni, A20⁺ mut16subser6 Ser sub 178 4b ni, no capsidmut19subser7 Ser sub 224 4b ni, no capsid mut32subser8 Ser sub 454 4bni, no capsid mut37subser9 Ser sub 526 4b ni, no capsid mut39subser10Ser sub 553 4b ni, no capsid mut40subser11 Ser sub 562 4b ni, no capsidmut41subser12 Ser sub 590 4b ni, no capsid mut44subser13 Ser sub 638 4bni, no capsid mut45subser14 Ser sub 664 4b ni, no capsid mut46subser15Ser sub 682 4b ni, no capsid mut4subflg2 FLAG sub  39 4a ni, A20⁺mut8subflg3 FLAG sub  76 4a ni, A20⁺ mut16subflg4 FLAG sub 178 4a ni,A20⁺ mut32subflg5 FLAG sub 454 4a ni, A20⁺ mut37subflg6 FLAG sub 526 4ani, A20⁺ mut38subflg7 FLAG sub 547 4a ni, A20⁺ mut40subflg8 FLAG sub 5624b ni, no capsid mut44subflg9 FLAG sub 638 4b ni, no capsidmut45subflg10 FLAG sub 664 4b ni, no capsid mut46subflg11 FLAG sub 6824b ni, no capsid^(a)Superscripts 1 to 4 indicate that a restriction site was introducedas a result of the alanine substitution mutation: 1, NheI; 2, EagI, 3,HpaI; 4, MluI.^(b)Ala sub, alanine substitution mutant; Ala ins, string of alanineresidues inserted after the indicated amino acid; HA, AU, His, or Serins, insertion of the HA, AU, His, or Ser epitope immediately after theindicated amino acid of wt cap; Ser or FLAG sub, substitution of the Seror FLAG epitope for the wt AAV capsid sequence beginning immediatelyafter the indicated# AAV amino acid residue. Amino acid tags: HA, YPYDVPDYA (SEQ ID NO:36); AU, DTYRYI (SEQ ID NO: 37); His, HHHHHH (SEQ ID NO: 38); Ser, FVFLI(SEQ ID NO: 39); FLAG (SEQ ID NO: 40), DYKDDDDK (SEQ ID NO: 41).^(c)pd, partially defective for infectivity, between 1 to 3 logs lowerthan wt; cs and hs, cold sensitive and heat sensitive, respectively; ni,noninfectious, 5 logs lower than wt; hep⁺, mutant bound to a heparancolumn; hep⁻, mutant did not bind to heparan sulfate; no capsid, mutantwas A20 ELISA negative and MAb, B1 negative; A20⁺, mutant could bedetected with A20 antibody;# surface, position was present on the surface of the capsid.^(d)The serpin insertion in VPN2 was KFNKPFVFLI (SEQ ID NO: 42).5.3.2.2 Infectious Titer Assays Reveal Four General Classes of Mutants

To determine the effect of each mutation on viral infectivity, either wtpIM45 or a mutant pIM45 plasmid was used to complement the growth ofpTRUF5. pTRUF5 is a recombinant AAV plasmid that contains the gfp geneunder the control of a CMV enhancer-promoter (Klein et al., 1998). Theresulting recombinant TRUF5 virus contained either wt or mutant capsidproteins and could be tittered for infectivity by counting greenfluorescent cells in the presence of an Ad5 coinfection. It had beenshown previously that the fluorescent cell assay produced titers withintwo- to threefold of those obtained with a conventional infectiouscenter assay (Zolotukhin et al., 1999). Initially, each mutant was grownand tittered at either 39.5° C. or 32° C. to determine if any of themutants were temperature sensitive. The studies were performed twice,and there was no significant variation in titer. On the basis of thesetiters, the mutants could be grouped into four classes (FIG. 9; Table7). Class 1 contained mutants that have an infectious titer similar tothe wt titer (less than 1 log difference; for example, mut1 and mut2).Class 2 contained partially defective mutants with infectious titers 2to 3 logs lower than the wt titer (for example, mut4 and mut5). Class 3contained temperature-sensitive mutants; three of these (mut26, mut27and mut33) were heat sensitive, and two (mut28 and mut35) were coldsensitive. Class 4 consisted of 12 noninfectious mutants, whose titerswere more than 5 logs lower than the wt titer.

5.3.2.3 (Class 4) Mutants and Temperature-Sensitive (Class 3) Mutantswere Defective in Packaging DNA or in Forming Stable Virus Particles

To determine the probable causes for the different defective mutants,attention was first given to class 3 and 4 mutants. For convenience, thefact that the temperature-sensitive mutants had now infectivity whengrown at the partially restrictive temperature of 37° C. was ignored,and viral preparations for all class 3 and 4 mutants were made at 37° C.To determine if these mutants were able to make capsids, an A20 ELISAwas used. The A20 antibody recognizes only intact AAV particles (Wistubaet al., 1997) and is useful for determining the physical particle titerirrespective of whether the capsids contain DNA (Grimm et al., 1999).Eight of sixteen mutants that were tested were negative by ELISA reading(Table 8), indicating that they were unable to make capsids or that thecapsids were unstable even in crude lysate preparations. All of theseclass 4 (noninfectious) mutants were classified as class 4b (Table 7;FIG. 8). TABLE 8 DETERMINATION OF PHYSICAL PARTICLE TITER ANDDNA-CONTAINING PARTICLE TITER OF CLASS 2 AND 3 MUTANTS Construct^(a) A20ELISA^(b) QC-PCR ™^(c) pIM45 (wt) +++ +++ mut19 − − mut20 − − mut22 ++++ mut23 − − mut24 − − mut25 − − mut26 (hs) ND^(d) ND mut27 (hs) + NDmut28 (cs) + ND mut31 ++ − mut33 (hs) ++ + mut35 (cs) ++ ++ mut37 ++ +mut40 ++ ++ mut42 − − mut46 − ND mut47 − ND^(a)hs, heat sensitive; cs, cold sensitive.^(b)+++, >10¹² particles/ml; ++, >10¹¹ particles/ml; +, >10¹⁰particles/ml; −, <10⁸ particles/ml, which was the limit of detection byA20 ELISA.^(c)+++, >10¹¹ full particles/ml; ++, >10¹⁰ full particles/ml; +, >10⁹full particles/ml; −, <10⁸ full particles/ml.^(d)ND, not done.

QC-PCR™ assays also were performed on most of the class 3 and 4 mutants.The QC-PCR™ assay measures the titer of AAV particles that containDNase-resistant rAAV genomes. It has been shown previously that itprovides physical particle titers that are equivalent to those obtainedby dot blot assay but has better sensitivity at low particle titers(Zolotukuhin et al., 1999). As expected, mutants that were negative forthe synthesis of AAV particles by A20 ELISA were also negative byQC-PCR™ assay (Table 8). Most of the remaining mutants, which werepositive for A20 particles, were also positive for packaged viral DNA inthe QC-PCR™ assay (Table 8). This group of noninfectious mutants (mut22and mut37) were called class 4a (Table 7; FIG. 8). Their defect was notin packaging but rather in the binding, internalization, or uncoatingsteps of the viral entry process. One A20-positive mutant (mut31) was anexception in that it was A20 positive but DNA negative by QC-PCR™ assay.This meant that mut31 formed intact virus particles that were empty. Toconfirm this, mut 31 was examined by EM, and it did indeed make emptyparticles. In contrast, the partially defective class 2 mutant, mut4,produced particles similar to wt particles. mut31 was assigned to class4c (Table 7).

5.3.2.4 Some Mutants are Defective for Binding the Viral Receptor

One potential cause for the reduced infectivity of class 2, 3 or 4mutants might be that they were unable to bind the viral cell surfacereceptor, the first step of the infectious cycle. Heparan sulfateproteoglycan has been identified as the primary cell surface receptorfor AAV (Summerford and Samulski, 1998). To test whether these mutantscould bind heparan, a heparan column binding assay was developed.lodixanol-purified wt or mutant rAAVs were passed through a heparanagarose column, and the AAV capsid proteins in the starting material andthe bound (eluate) and unbound (flowthrough and wash) fractions weremonitored by Western blotting using MAb B1, which recognizes all threecapsid proteins (Table 9). As expected, wt AAV had a high affinity forthe heparan column, since little capsin protein was detected in theflowthrough and wash fractions, and most of the capsid protein wasdetected in the eluate. The same was true of most of the mutants tested(Table 9). Two mutants, however, mut35 and mut41, bound poorly toheparan. A third mutant, mut40, which is located about 20 aa away frommut41, also bound with reduced affinity. This suggested that the primarydefect in these mutants was their inability to bind to heparan sulfateproteoglycan. Mut35 was classified as class 3b (temperature sensitiveand heparan binding negative), mut41 was classified as claim 2c(partially defective and heparan binding negative), and mut40 wasclassified as class 4d (noninfectious and heparan binding negative)(Table 7). TABLE 9 HEPARAN COLUMN BINDING PROPERTIES OF CLASS 2, 3 AND 4MUTANTS^(a) Construct Heparan Binding pIM45 + mut4 + mut5 + mut6 +mut7 + mut8 + mut10 + mut11 + mut12 + mut13 + mut14 + mut15 + mut18 +mut20 0 mut21 0 mut22 + mut25 0 mut27 0 mut28 + mut30 + mut31 + mut32 +mut33 + mut34 + mut35 − mut36 + mut37 + mut39 0 mut40 − mut41 − mut43 +mut46 0 mut48 +^(a)+, mutant virus bound to a heparan column with the same affinity aswt pIM45 virus; −, virus bound with at least a threefold-lower affinity;0, no protein signal detected by Western blotting.

Three class 4b mutants, mut20, mut25 and mut46, could not be detected byWestern analysis (Table 9). This was consistent with the fact that theymade no capsid that was detectable with the A20 antibody (Table 8).Additionally, mut27, a temperature-sensitive mutant, and two class 2mutants, mut21 and mut39, did not give any Western signal with MAb B1(Table 9). The heat-sensitive mutant, mut27, was presumably unstable atthe nonpermissive temperature used for growing this virus. mut21 andmut39 were partially defective when assayed in crude extracts. The factthat they could not be detected by capsid antibody after iodixanolcentrifugation suggests that these capsids were also unstable duringpurification. These mutants were assigned to class 2b on the basis oftheir capsid instability (Table 7). The rest of the mutants in class 2that bind to heparan were classified as class 2a, partially defective,and heparan binding positive (Tables 7 and 9). The nature of theirdefect was not clear but presumably was due to some step in theinfectious process that occurs after viral attachment to the cellsurface.

5.3.2.5 Regions Tolerating Alanine Substitutions do not Tolerate OtherKinds of Substitutions

It was wanted to determine whether the class 1 mutants defined positionsin the capsid genes that were truly nonessential for capsid function. Totest this, a series of mutants were constructed in which either theserpin receptor ligand, FVFLI (SEQ ID NO:43) (Ziady et al., 1997), orthe FLAG antibody epitope, DYKDDDDKYK (SEQ ID NO:44), was substitutedfor capsid sequences at many of the class 1 mutant positions (Table 10).A number of class 2 and class 4 mutants were tried as well. The serpinsubstitution (5 aa) was the same size as the largest alaninesubstitutions. The FLAG epitope is highly charged, as were many of thesubstituted wt sequences. As expected, substitutions at class 2(partially defective) or class 4 (nonviable) positions did not produceinfectious virus (Table 10). Surprisingly, although many of the class 1serpin or FLAG substitutions produced some physical particles detectablewith the A20 antibody, only one of the substitutions, serpin at aa 34(the mut3 position), produced infectious virus particles in substantialyield (Table 10). Most infectious titers were reduced by 5 logs or more,and particle titers (as judged by A20 ELISA) were reduced orundetectable as well. Thus, although modification of charged residues inclass 1 mutants to alanine was permissible, these regions of the capsidwere nevertheless essential for capsid formation and were sensitive toother kinds of substitutions. TABLE 10 SUBSTITUTION OF SERPIN OR FLAGEPITOPES AT CAPSID POSITIONS THAT TOLERATED ALANINE SUBSTITUTIONSTiter^(a) Mutant Infectious Physical Particle mut1subser1 − +mut2subser2 − + mut3subser3 1 log lower + mut9subser4 − + mut14subser5− + mut16subser6 − − mut19subser7 − − mut32subser8 − − mut37subser9 − −mut39subser10 − − mut40subser11 − − mut41subser12 − − mut44subser13 − −mut45subser14 − − mut46subser15 − − mut4subflg2 − + mut8subflg3 − +mut16subflg4 − + mut32subflg5 − + mut37subflg6 − + mut38subflg7 − +mut40subflg8 − − mut44subflg9 − − mut45subflg10 − − mut46subflg11 − −^(a)Either a serpin peptide sequence or the FLAG sequence wassubstituted for the AAV capsid sequence at the positions used previouslyfor alanine scanning mutagenesis (FIG. 14). Infectious titers weredetermined by GFP fluorescent cell assay. −, infectious virus could notbe detected. Physical particle titers were judged by A20 ELISA. +,particles were detectable; −, particles were not detectable.5.3.2.6 Putative Loop Regions and the N-Terminal Regions of VP1 and VP2are Able to Accept Insertions of Foreign Epitopes

Several other sites were also chosen for insertion of foreign sequences.For these mutants, the less charged HA epitope, YPVDVPDYA (SEQ IDNO:45), was inserted. The target positions for insertion were theN-terminal regions of the three capsid proteins, VP1, VP2 and VP3, the Cterminus of the cap ORF and seven positions (mutants L1 to L7) that werebelieved to be in loop regions of the capsid protein based on analignment of the AAV capsid sequence to that of CPV (Chapman andRossman, 1993). Since these sites were suspected to be on the surface ofthe capsid, insertions at these sites might not affect capsid assemblyor stability. Mutations in the loop regions had been targetedsuccessfully before by Girod et al. (1999), who were able to insert theL14 ligand at aa 587 without significant loss in infectivity.

Insertions at the N termini of VP1 (VPN1) and VP3 (VPN3) and the Cterminus of the cap ORF (VPC) were not well tolerate (Table 11). Toeliminate the possibility that the defect in these mutants was due tothe HA tag, other tags, such as AU, His, and Myc, were also inserted atthe N termini of VP1 and VP3 and the C terminus of cap, and they alsowere not tolerated at those positions (Table 7). Insertions at three ofthe putative loop regions were also not viable (Table 11, mutants L2, L4and L5). Mutants L4 (aa 522) and L5 (aa 553) were interesting in thatthey produced a significant yield of physical particles that were notinfectious. TABLE 11 HA INSERTION MUTANTS Titer Mutant PositionInfectious^(a) Physical Particle^(b) L1 aa 266 ++ + L2 aa 328 − + L3 aa447 ++ ++ L4 aa 522 − ++ L5 aa 553 − ++ L6 aa 591 ++ ++ L7 aa 664 ++ ++VPN1 aa 1 + ++ VP1 aa 34 +++ ++ VPN2 aa 138 +++ +++ VPN3 aa 203 − − VPCC terminus − −^(a)Determined by GFP fluorescence cell assay. +++, 1 log lower than wt;++, 2 logs lower; +, 3 lots lower; −, at least 5 logs lower.^(b)Determined by A20 ELISA. +, 4 logs lower than wt pIM45; ++, 2 to 3logs lower; +++, 1 log lower; −, undetectable.

However, HA insertions were well tolerated at aa 34 within theN-terminal region of VP1, at the N terminus of VP2, and within three ofthe putative loop regions, loop I (mutant L1), loop IV (mutants L3 andL6), and loop V (mutant L7) (Table 11).

5.3.2.7 Some HA Insertion Positions are on the Capsid Surface

To determine whether the HA insertion mutants contained the HA sequenceexposed on the surface of the capsid, batch immunoprecipitation with HAMAb-conjugated beads was used. In each case, virus was purified byiodixanol density centrifugation and heparan column chromatography toremove any soluble capsid protein that might be present in crude viralpreparations. As expected, insertion of the HA tag at the N terminus ofVP2 (mutant VPN2) produced a slight increase in the molecular weight ofVP2 and VP1 compared to wt protein, pIM45. In the case of the VP1 mutant(HA insertion at aa 34 in VP1), only VP1 had a higher molecular weightand only VP1 contained the HA tag, as expected. When the viableinsertions, VPN2 (HA insertion at the N terminus of VP2) and VP1(insertion at aa 34), were treated with HA MAb-conjugated beads,substantial amounts of both viruses were precipitated. This demonstratedthat in both cases the HA epitope was on the surface of the virusparticle and accessible to the antibody. Control wt virus particles,were not precipitated with HA MAb to any significant extent. The amountof virus in the starting material was monitored by Western blotting withB1 or HA MAb.

The putative loop HA insertion mutants, L1 to L7, were also incubatedwith HA MAb-conjugated beads. Although the insertions in some of thesemutants produced noninfectious virus, they all produced sufficient A20antibody-positive virus particles to test for the presence of the HA tagon the surface of the capsid. When this was done, all of the L-seriesinsertions were shown to be in the immunoprecipitate (bound fraction)compared to the wt (pIM45) control. This demonstrated that each of thoseinsertions at putative loop sites resulted in the HA epitope being onthe surface of the capsid.

Interestingly, two loop insertion mutants, L4 and L6, were found to bindheparan columns with reduced affinity, which probably accounted for thelower infectivity of these mutants in the standard fluorescent cellassay. The L4 and L6 insertions were near the heparan-binding-negativemutants mut35, mut40, and mut41. All five of theseheparan-binding-negative mutants were located between aa 509 and 591,suggesting that this region within the AAV capsid constitutes theheparan binding domain of the capsid protein.

5.3.2.8 Changing the Tropism of AAV

To determine whether the tropism of rAAV could be changed by inserting anovel receptor ligand into the capsid, two mutant plasmids wereconstructed that contained a serpin receptor ligand. In one case, theserpin ligand FVFLI (Ziady et al., 1997) was substituted for the AAVcapsid sequence immediately after aa 34. In the second mutant, anexpanded serpin receptor ligand, KFNKPFVFLI (SEQ ID NO:46) (Ziady etal., 1997), was inserted at the N terminus of VP2, aa 138 (Table 7). Themutant capsid plasmids were then used to package CBA-AT, an rAAV genomethat contained the hAAT gene under the control of a hybrid CMV-β-actinpromoter. As seen with the HA insertion mutants described above, theserpin mutants produced rAAV viral titers that were slightly (six-fold)lower in infectivity when tittered by the infectious center assay on 293cells. However, when equal amounts of wt or mutant virus (as determinedon 293 cells) were infected into IB3 cells, both mutant viruses showedsubstantially higher infectivity than wt (FIG. 10). The VP2 serpininsertion was 15-fold more infectious, and the VP1 substitution mutantwas approximately 62-fold more active. This suggested that IB3 cells, alung epithelial cell line believed to express the serpin receptor, werea much better target for the serpin-tagged chimeric rAAVs than wt andthat the tropism of the mutant rAAVs had been changed. Because bothmutants retained the wt heparan binding region, IB3 cells were alsoinfected in the presence of heparan sulfate to see if they continued touse heparan sulfate proteoglycan for viral entry. When this was done,both wt and mutant infectivity dropped to barely detectable levels (FIG.10). Taken together, these findings suggest that the serpin-taggedviruses continued to use heparan sulfate proteoglycan as the primaryreceptor and were using an alternative co-receptor, presumably theserpin receptor.

5.3.3 Discussion

In this study, the phenotypes of 93 AAV2 capsid mutants at 59 differentpositions within the capsid ORF are described. Several classes ofmutants were analyzed, including epitope tag or peptide ligand insertionmutants, alanine scanning mutants, and epitope substitution mutants.From this, some eight separate phenotypes could be identified (Table 7).

5.3.3.1 Noninfectious Mutants

The bulk of the mutants that were noninfectious either were unable toassemble capsids or the capsids were unstable. These mutants (class 4b)were located predominantly but not exclusively in which are likely to beβ-strand structures in the capsid proteins. Two of these mutants wereinsertions at the N- and C-terminal residues of VP3, suggesting thatboth ends of VP3 play a role that is important for capsid assembly orstability. Ruffing et al. (1994) have previously characterized deletionsof the C terminus of the capsid ORF, and these deletions also werenoninfectious.

One noninfectious mutant, mut31, produced viable capsids that wereempty. Thus mutant, which consists of a single amino acid substitution(R432A), was apparently defective in packaging viral DNA and is locatedin putative loop IV. It is not clear what the mechanism of viral DNApackaging is. Ruffing et al. (1992) demonstrated that empty capsidscould assemble in the absence of viral DNA. Some studies have suggestedthat packaging is an active process that requires interaction of Repproteins with capsid proteins (Weger et al., 1999) or possibly iscoupled with DNA replication (Zhou and Muzyczka., 1999).

Most of the remaining noninfectious mutants (class 4a) were capable ofassembling capsids and packaging DNA. These are likely to be defectivein some aspect of viral entry or uncoating and will require furtherstudy to uncover the mechanism of the defect.

5.3.3.2 Receptor Binding Mutants

Two of the noninfectious mutants, mut40 and L4, were apparentlynoninfectious because they were unable to bind to heparan sulfate (class4d). Heparan sulfate proteoglycan is believed to be the primary cellsurface receptor for AAV (Summerford and Samulski, 1998). Three othermutants also were identified as defective for binding heparan sulfate,two partially defective mutants (class 2c), and onetemperature-sensitive mutant (class 3b). Together, the five mutants weredistributed into two clusters in loop IV that were separated by 40 aa.The first cluster spanned aa 509 to 520 (mut35 and L4); the second wasbetween aa 561 and 591 (mut40, mut 41 and L6). Mutants L4 and L6consisted of HA epitope insertions into the two heparan bindingclusters. These were found to be capable of being immunoprecipitated byHA MAb, confirming that these positions were on the surface of thecapsid. Girod et al. (1999) reported that insertion of the L14 epitopeat aa 587, the position of the heparan-negative mut41 mutant, wascapable of targeting the virus to the L14 receptor, thus confirming thatthis region is on the surface of the capsid. A heparan-negativeinsertion mutant also was reported by Rabinowitz et al. (1999) whilethis report was in preparation; it fell near the cluster at aa 522.Taken together, analyses of these mutants suggest that the putative loopIV region contains two blocks of residues that are on the surface of thecapsid and involved in heparan sulfate binding.

A heparan binding motif which consists of a negatively charged aminoacid cluster of the type XBBBXXBX (SEQ ID NO:47) (where B is a basicamino acid and X is any amino acid) has been identified in severalreceptors and viruses (Hileman et al., 1998). Regions containing theseclusters also appear to be sensitive to spacing changes. Although noheparan binding consensus motif of this kind was found in a variety ofheparan binding mutants, there were basic amino acids near thesedomains. mut35, an insertion at aa 509, was near basic amino acids K507and K509. Interestingly, K507 is conserved in AAV1, −2, −3, −4, and −6and in AAV5 is an R. H509 is present only in AAV2 and −3. AAV1, −2, and−3 are known to bind to heparan sulfate, while AAV4 and −5 do not.Additionally, L4, an insertion at aa 520, was near basic amino acidsR585 and R588. H526 and K527 are conserved except for AAV4 and −5, whileR585 and R588 are unique to AAV2. For all of these mutants, theinsertions could have disrupted local conformation that hindered normalheparan binding. For mut41, R-to-A substitutions at aa 585 and 588 mightcontribute directly to reduced heparan binding. Finally, mut40 did notaffect either basic amino acids or spacing within the capsid protein.

5.3.3.3 Capsin Regions that are on the Surface of the Virus Particle

In addition to the heparan binding clusters, several other regions werealso present on the capsid surface. These include four of the fiveputative loop regions (mutants L1 to L7), the N terminus of VP2 (mutantVPN2), and a region within the N terminus of VP1 at amino acid 34(mutant VP1). HA epitope insertions at these positions were all capableof being immunoprecipitated with anti-HA antibody. The L1 insertionmutant at aa 266 had the peculiar phenotype of being partially viable(Table 7) but was not detectable with the A20 MAb, an antibody thatrecognizes a conformational epitope that is present only in intact viralparticles. A nearby capsid forming mutant made by Girod et al. (1999) ataa 261 was also negative for A20 antibody binding. This suggests that atleast part of the epitope for the A20 MAb consists of amino acidsbetween 261 and 266 and confirms that this region is on the surface ofthe intact particle.

Of the positions identified as being on the surface of the capsid, sixwere identified that potentially are capable of accepting foreignepitope or ligand insertions for retargeting the viral capsid toalternative receptors. These are the N-terminal region of VP1 (near aa34), the N terminus of VP2 (aa 138), the loop I region (aa 266), theloop IV region (near aa 447 and 591), and the loop V region (aa 664).All of these locations were capable of tolerating an HA (or serpin)insertion and produced recombinant virus titers that were within 1 to 2logs of the wt value. Furthermore, HA epitope insertions at thesepositions were capable of being immunoprecipitated with anti-HAantibody. Two of these positions, when tested with a serpin ligandinsertion or substitution, produced virus that was much more infectiouson IB3 cells than wt virus. Curiously, both serpin mutants were stillinhibited by soluble heparan sulfate, suggesting that heparan sulfateproteoglycan was still the primary receptor for these mutants and thatthe serpin receptor was being used as an alternative co-receptor. It isconceivable that one or both of these capsin positions is involved inbinding to one or both of the proteins that normally act as co-receptorsfor wt virus, fibroblast growth factor (Qing et al., 1999), or integrinα_(v)β₅ (Summerford et al., 1999). This would explain their partialdefect on 293 cells and the recovery of infectivity on IB3 cells.

5.3.3.4 Mutants with Unstable Capsids and Temperature-sensitivePhenotypes

Three mutants, mut21, mut27 and mut39, were found to have capsids thatwere unstable when purified through an iodixanol gradient. Iodixanol isan iso-osmotic gradient purification method that appears to be gentlerthan CsCl centrifugation (Zolotukhin et al., 1999). Thus, these mutantsappear to be particularly sensitive to capsid denaturation. mut21 andmut27 are in putative β sheets, and mut39 is in loop IV. It is worthnoting that Rabinowitz et al. (1999) also isolated an unstable capsidmutant at aa 247 that is near the mut21 position, aa 254. mut27 is alsoone of five temperature-sensitive mutants isolated during this study.

5.3.3.5 Viable and Partially Defective Mutants

The two largest classes of mutants isolated with either wt (class 1) orpartially defective (class 2a) with no identifiable defect. Both class 1and class 2a mutants were distributed either in the VP1 and VP2 uniqueregions or in the predicted loop regions of the capsid protein. It wasnaively assumed that class 1 mutant positions, which produced viablecapsids after substitution of two to five alanine residues, were regionsthat were nonessential for capsid assembly or stability and thereforeshould accommodate other kinds of substitutions. However, when serpin orFLAG epitopes were substituted at many of these sites, most of themutants were nonviable, with the exception of aa 34 in VP1. Indeed, manyof these viruses were negative for capsid assembly and should also beuseful for identifying possible intermediates in capsid assembly.

Ruffing et al. (1992) showed previously that VP1 and VP2 but not VP3contained nuclear localization signals (NLS), and three putative NLS arelocated in the VP1/VP2 region at aa 121 to 125, 141 to 145, and 167 to171. Hoque et al. (1999) have shown that aa 167 to 172 were sufficientto target VP2 to the nucleus, although their experiments did not ruleout possible redundancy with the other two putative NLS sequences. Allthree of these putative signals were targeted with alanine scanningmutants (mut12, mut13 and mut15). Two of these mutants, mut12 and mut15,were partially defective, and the inactivation of an NLS may be thereason for their phenotype (Hoque et al., 1999; Ruffing et al., 1992).mut15 should have eliminated the NLS identified by Hoque et al. The factthat mut15 was only partially defective suggests that there may be analternative, redundant NLS sequences that are used by the capsidproteins. The third mutant (mut13) was classified as viable, but it alsoshowed a lower than wt titer.

5.3.3.6 Molecular Computer Graphics Construction of an AAV Model andStructural Localization of Mutant Residues

Because the AAV crystal structure is not available, the atomiccoordinates of CPV VP2 (PDB accession No. 4DPV) were interactivelymutated using the program O (Jones et al., 1991) to generate ahomology-based model of the AAV capsid, using modifications of thealignments of the AAV major capsid protein (VP3) with the VP2 capsidprotein of CPV (Chapman and Rossman, 1993; Girod et al., 1999). Themutations were followed by refinement constrained with standard geometryin the O database. The model provided a means for preliminary structuralidentification of the heparan receptor attachment sites in the surfacedepression (dimple) near the twofold icosahedral axes of the capsid,surface loop regions which can tolerate foreign peptide insertions, anda possible explanation for the phenotype of mut31 (FIG. 11).

The topographic location of the putative heparan binding region isconsistent with regions that have been suggested as being involved inhost cellular factor(s) recognition and implicated in tissue tropism andin vivo pathogenicity for other parvoviruses (Agbandje-McKenna et al.,1998; Barbis et al., 1992; McKenna et al., 1999; Tresnan et al., 1995).It is of interest that the putative heparan binding site is adjacent toa region of the AAV capsid that contains a peptide insert when the AAVVP3 sequence is compared to that of CPV VP2 and the VP2 of most of theother autonomous parvovirus sequences (Chapman and Rossman, 1993). Also,a similar insertion of peptide sequences compared to CPV (although notin a homologous region of the VP2 to that observed in AAV) is present inthe capsid of Aleutian mink disease parvovirus and minute virus of mice,proximal to residues in the dimple depression which are implicated intissue tropism (McKenna et al., 1999). Thus, these insertions may becapsid surface adaptations that enable the capsids to recognizedifferent receptors during infection. In the case of AAV, its dimplepeptide insertion, which is absent in the other parvoviruses, may enableit to recognize heparan sulfate, which ahs not been implicated incellular infectivity by any other parvovirus.

The model also clearly shows that regions of the capsid which toleratedinsertions of the HA epitope (ie., at residues 266, 447, 591 and 664)are on the surface loops present between the β strands of the β-barrelmotif (FIG. 11). The β-barrel motif forms the core contiguous shell ofparvovirus capsids, while the surface loops make up the surfacedecorations, dictating the strain-specific biological properties of themembers. The observation that these surface regions can tolerate foreignpeptide insertion is an indication that they are not involved in theinteractions that govern capsid assembly.

Finally, the model provides a possible explanation for the observationthat mut31 (R432A) I able to form only empty particles. In theunassembled VP3 monomer, the side chain of R432, points toward theinterior of the capsid and would most likely be in contact with DNA. Ifrecognition and encapsidation of AAV DNA precede final capsid assemblyand involve oligometric intermediates, then R432 contacts with DNA maybe essential for initiating capsid assembly around a nascent DNA strand.

5.4 Example 4

Transduction of Human Islets of Langerhans

In order to determine whether human islets were permissive fortransduction with rAAV vectors, a series of transduction experimentshave been performed with islets provided by the University of Miamiislet cell isolation core. Initially, both the rAAV-CMV-GreenFluorescent Protein (GFP) vector, UF5, or the rAAV-CMV/β-actin hybridpromoter (CB)-GFP vector, UF11 have been used. In these studies, batchesof approximately 1000 islets were infected in 4.84 cm² slide chambers ata multiplicity of infection (MOI) of approximately 10,000 infectiousunits (i.u.) per cell or 1,000. Lower MOIs failed to show any evidenceof expression. Cells were co-infected with Ad5 (MOI of 5) to accelerateleading strand synthesis in these short-term experiments (although thishas never been necessary in vivo if one is able to tolerate a 2 to 4week delay before maximal expression). Expression was quite efficient inislets 48 hr after infection under these conditions. Interestingly,transduction was much less efficient at an MOI of 1000, and wasundetectable at an MOI of 100 or less.

In order to confirm that β cells within the islet were transduced inthese experiments, a similar batch of islets was cytocentrifuged, fixed,and immunostained with a TRITC-conjugated anti-insulin antibody. Cellswere then examined by fluorescence microscopy to determine whether thered β cell label co-localized with the green GFP fluorescence. Clear-cutexamples of co-localization were frequently observed, indicating thatthis important cell type had been transduced.

5.4.1 Modification of AAV Capsid Proteins to Facilitate Cell Targeting

Scanning mutagenesis of the AAV capsid proteins, VP1, VP2, and VP3 hasbeen performed, and several sites have been characterized in whichpolypeptide insertions have been tolerated without loss of capsidstability or integrity (DNase resistance). In order to determine whetherthe trasnsduction of a relatively nonpermissive cell type (bronchialepithelium) could be enhanced, the rAAV vector, CB-AT (expressing humanα1-antitrypsin from the CB promoter) was packaged into each of threecapsid types: wild-type (unmodified) capsid, capsid with a peptide(FVFLI or KFNKPFVFLI) (SEQ ID NO:48) ligand for the secR (referred tohere as “secRL”) inserted internally at residue 34 of VP1, or capsidwith the same secRL inserted at the amino-terminus of VP2. The CFbronchial epithelial cell line, IB3-1 was infected with each intriplicate (1.5×10⁵ cells per 15 mm-diameter well) at an MOI ofapproximately 400 i.u. per cell, either in the presence or absence ofsoluble heparin, 2 mg/ml. The VP1-34 serpin containing capsid mediated asignificantly higher level of hAAT expression than with wild-typecapsid, with the VP2N serpin being intermediate. Interestingly, theseeffects were more pronounced in the absence of soluble heparin sulfate,although the capsid insertions did show a two- to three-fold enhancementover wild-type even in the presence of soluble heparin sulfate. Theseresults suggest that these capsid modifications can either enhance entrythrough alternative, non-heparin dependent, pathways or they cansynergize with heparin to greatly enhance vector attachment and entry.

5.4.2 Experimental Methods

5.4.2.1 Design

The luciferase (luc) and green fluorescent protein (GFP) reporter geneswere used for these studies. Luc is primarily used for all comparativestudies of promoters and conditions, while GFP is used under optimalconditions to gauge what percentage of cells is transduced at anycorresponding level of luciferase activity. The primary luciferasevectors used in these studies are shown in FIG. 12.

Cell culture wells (4.84 cm²) containing either 1000 human islets, 500murine islets, or 5×10⁵ endothelial cells are infected with eachconstruct and assayed for transgene expression at 48 hrs. Initialendothelial studies employ the ECV304 endothelial cell line (Takahashiet al., 1990, which has been used extensively as a model forendothelium, although some phenotypic features are not preserved (Brownet al., 2000). Positive results are verified by testing expression inhuman umbilical vein endothelial cells (HUVECs). In parallel islet cellor endothelial cell cultures handled without Ad augmentation, replicatewells are assayed at time points up to 10 days, since published resultsindicate that vector expression is comparably efficient although delayedby several days under those conditions (Afione et al., 1999). Luciferaseis assayed by luminometry using a commercially available kit, while GFPexpression is evaluated by fluorescence microscopy on either ZeissAxioskop or by confocal imaging. Images are processed with a Metamorphimaging package. After an initial comparison of the CMV, CB, elongationfactor la (EF) and insulin promoters with luciferase constructs (FIG.12), selected promoters are re-evaluated with GFP vectors to score forpercent transduction.

5.4.2.2 Efficiency of Transduction of Target Cell Types Assessed Ex Vivo

As indicated, two sites within the capsid have been identified, at aminoacid 34 of VP1 (VP1-34) and at the extreme N-terminus of VP2 (VP2N),that will tolerate insertion of new epitopes, both maintaining theviability of the virion and presenting these new epitopes on the capsidsurface in a position accessible to antibody binding and cell binding.Comparison with the predicted structure of the AAV capsid proteinsindicates that these residues would likely be on the outside of thevirion.

Several cell surface receptors have been identified in endothelial cellsthat may possibly be used for targeting recombinant AAV to endothelialcells and/or islet cells. Endothelial cells bind acetylated LDL (Steinand Stein, 1980; Voyta et al., 1984). Acetylated LDL uptake is efficientin hepatic endothelial cells (Pitas et al., 1985), primarily by thescavenger receptor class B type 1 (SR-B1) receptor (Acton et al., 1994;Varban et al., 1998). This receptor, however, is notendothelial-specific; it is expressed at high levels in fat (Acton etal., 1994) macrophages (Hirano et al., 1999), and steroidogenic tissues(Cao et al., 1999), and at significant levels in vascular smooth musclecells (Mietus-Snyder et al., 1998), fibroblasts (Pitas, 1990), and othercell types, but is expressed less efficiently in the kidney (Acton etal., 1994). Recent work by Grupping et al. (1997) has demonstrated thatthis receptor is present and functional on β cells of the islet as well.A minimal polypeptide sequence of 28 amino acids (referred to here as“ApoEL”; consisting of LRKLRKRLLR [SEQ ID NO:1] from hApoE+thelipid-associating peptide DWLKAFYDKVAEDLDEAF [SEQ ID NO:21]) has beenshown to be efficient for binding to the LDL-R and stimulating itsinternalization (Datta et al., 2000). This would be within the sizerange of ligands previously tolerated within either the VP1-34 or VP2Nsites.

E-selectin is another potential receptor for targeting recombinant AAVto endothelium. E-selectins are calcium-dependent receptors for sialylLewis carbohydrate moieties on the plasma membrane of leukocytes thatcause them to adhere to vascular endothelium (Vestweber and Blanks,1999). Endothelial E-selectin expression is induced by inflammatorycytokines, such as TNF-α and IL-1, by interaction of CD40 withendothelium (Pober, 1999), but is also expressed in proliferatingendothelial cells in the absence of inflammation (Luo et al., 1999).Recently a small peptide ligand (referred to here as “SelL”; consistingof IELLQAR (SEQ ID NO:49) was identified by phage display. This peptidebinds tightly to E-selectin and inhibits the binding of sialyl Lewis Xor sialyl Lewis A oligosaccharides to E-selectin (Fukuda et al., 2000).By incorporating this peptide sequence into the N-terminal VP2 site, itmay be possible to target AAV constructs specifically to endothelium atsites of inflammation or endothelial proliferation. In addition, morewidespread delivery of rAAV may be induced by infusion of quantities ofIL-1 sufficient to produce limited expression of E-selectin (Wyble etal., 1997).

In order to determine whether these capsid modifications will facilitaterAAV infection of islets, each of these capsid inserts have beenengineered into the pIM45 backbone. This is an ITR-deleted (ort) AAVRep/cap-expression helper construct. A candidate highly activeconstitutive rAAV reporter gene vector may be selected by tripletransfection of the vector (e.g., pAAV-CMV-luc), the Ad helper geneplasmid pXX6, and the new AAV helper (VP1-34ApoEL or VP2NApoEL).Wild-type AAV2 capsid (pIM45) is used to generate control vector virionsfor these experiments. This packaged material is then be tested fortransduction efficiency both in the presence and absence of solubleheparin sulfate (2 mg/ml) on each of the cell types (murine and human,1.5×10⁵ cells per 15-mm well) in the presence of Ad5, MOI of 10 (Table12). Each of these comparisons is performed in triplicate and therelative enhancement of short-term (48-hr. post-transduction) luciferaseexpression is assessed. TABLE 12 Murine Human Capsid/Cell Type MurineIslets Human Islets Endothelium Endothelium Wild-type (pIM45) N = 3 +hep, N = 3 − hep N = 3 + hep, N = 3 − hep N = 3 + hep, N = 3 − hep N =3 + hep, N = 3 − hep VP1-34-ApoEL N = 3 + hep, N = 3 − hep N = 3 + hep,N = 3 − hep N = 3 + hep, N = 3 − hep N = 3 + hep, N = 3 − hep VP2N-ApoELN = 3 + hep, N = 3 − hep N = 3 + hep, N = 3 − hep N = 3 + hep, N = 3 −hep N = 3 + hep, N = 3 − hep VP1-34-SelL — — N = 3 + hep, N = 3 − hep N= 3 + hep, N = 3 − hep VP2N-SelL — — N = 3 + hep, N = 3 − hep N = 3 +hep, N = 3 − hep

Promising combinations are then tested again in the presence of absenceof competing soluble LDL or with soluble E-selectin to confirm that theobserved effects are due to a specific reaction with the putativereceptor in question.

The N-terminal VP2 site could also potentially tolerate very largeinserts, or even single chain Fv antibodies directed against receptorsthat are known to be internalized when bound to ligand. This couldinclude either the LDL-R or the sulfonylurea receptor. A relatedstrategy will be employed for receptors where antibodies are availablein the form of traditional monoclonal or polyclonal antibodies.

5.4.2.3 Targeting Pancreatic Islets and Endothelial Cells in Vivo

5.4.2.3.1 Design

In order to determine whether it is possible to achieve in vivotransduction of pancreatic islets and renal vascular endothelium anintra-arterial injection protocol will be used in mice. The deliveryprotocol will be to cannulate the left common carotid and thread apre-measured catheter into the aortic arch and then into the descendingthoracic aorta, just rostral to the diaphragm for vector injections. Theabdominal aorta will be cross-clamped 1 cm below the diaphragm for 30sec during the injection. The same site of injection will be used forboth the islet cell and the endothelial transduction experiments, sincethe descending aorta just below the diaphragm is the source the bloodsupply to the kidneys (the renal arteries) and the blood supply topancreas (superior pancreatico-duodenal via the celiac artery andinferior pancreatico-duodenal via the superior mesenteric artery).

In initial studies designed to determine the efficiency of islet celltransduction, the vector backbone to be used is the insulin promoterdriving the human α1-antitrypsin (hAAT) cDNA. This work is done inC57Bl\6 mice, which have been shown to be tolerant to hAAT (Song et al.,1998). The advantage of using HAAT is that its expression can bemeasured serially over time in an individual animal by performing ahuman-specific AAT ELISA on small (10 μl) aliquots of serum obtainedfrom tail-bleeding. This ELISA has been used repeatedly to monitorexpression of hAAT in serum from mice injected with rAAV-hAAT vectors bythe intra-muscular, intra-portal, and intra-tracheal routes. The tissuespecificity of the insulin promoter permits one to determine whether theobserved hAAT expression is originating from the islets as opposed toother organs. In like fashion, an optimal endothelial-specific promoterchosen, e.g., the E-selectin promoter (Esel), is used in vector studiesdesigned to determine expression efficiency from the vascularendothelium. In each instance, comparison is made between rAAV-hAATvectors packaged in wt-AAV capsid and the same vector cassettes packagedin the receptor-targeted capsids (by the optimal genetic modificationand bi-specific antibody conjugations determined). Cohorts of 5 miceeach are injected with doses of either 3×10⁹ i.u. (low dose) or 3×10¹⁰i.u. (high dose) of vector intra-arterially, and hAAT levels aremeasured by serum ELISA at bi-weekly intervals for 6 months after theinitial injection. TABLE 13 rAAV-CB-hAAT Capsid/Vector rAAV-ins-hAATrAAV-Esel-hAAT (positive control) Wild type (pIM45) N = 5(hi dose) +5(lo dose) N = 5(hi dose) + 5(lo dose) N = 5(hi dose) VP-ApoEL N = 5(hidose) + 5(lo dose) N = 5(hi dose) + 5(lo dose) N = 5(hi dose) VP-SelL —N = 5(hi dose) + 5(lo dose) N = 5(hi dose) Islet receptor N = 5(hidose) + 5(lo dose) — — bispecific Endothelial bispecific N = 5(hidose) + 5(lo dose) —5.4.2.4 Regulation of Transcriptional Activity of the Vector InsertsUsing Tetracycline-Regulated Promoter, and/or Other Similar Systems

The therapeutic genes that are ultimately to be used in this programwill all have the potential for toxicity or other undesired effects ifthey are expressed at inappropriately high levels. Systems designed toregulate transcriptional activity have been well-established in vitroand in transgenic animal models, and earlier studies have shown thatthese systems could possibly be useful in vivo after gene transfer. Themethods disclosed may also be used to prepare constructs that utilizethe Clontech-Bujard tetracycline-regulated (tet) promoter system and theAriad ARGENT® system for delivery to pancreatic islets and renalvascular endothelium.

5.4.2.4.1 Design

Each of these is a two-component system. In the tet system, seven copiesof the tet operator sequence are engineered upstream of a minimal CMVpromoter to produce a tetracycline regulated element (TRE). The TRE isminimally active in the basal state. A transactivator protein, thereverse tetracycline transactivator (rtTA) is expressed from a secondgene. This protein consists of a mutant version of the DNA bindingdomain and ligand binding domain from the bacterial tet suppressor andthe transcriptional activation domain from the herpesvirus VP16 gene.The mutation allows the rtTA protein to bind to the TRE and activatetranscription only in the presence of doxycycline (a tetracyclinederivative). The ARGENT system is similar. The basic promoter (LH-Z₁₂-)requires transactivation by a dimerization dependent transcriptionalactivator that will dimerize only in the presence of the rapamycin-likedrug. Additional specificity can be conferred by expressing theappropriate trans-activator proteins from tissue specific promoters.

As with the earlier in vitro studies, the initial in vitro studies wereperformed with luciferase reporter constructs (rAAV-TRE-luc, andrAAV-LH-Z₁₂-luc) co-transduced into islet cell or endothelial cellcultures with either a CMV-driven or a tissue-specific promoter drivenversion of the appropriate transactivator. The level of luc expressionis assessed using standard methods over a range of concentrations of theinducing drug (Doxycycline or the Ariad dimerizer drug). Once theoptimal promoter choices for the transactivator genes are selected, thetransactivator gene and the inducible versions of the hAAT gene may becloned into single rAAV cassettes for in vivo applications.

In the in vivo studies, intra-arterial injections are performed byintra-arterial injection in C57Bl\6 mice. The most active version of thecapsid available at the time is then identified. After allowing 6 weeksfor completion of leading strand synthesis of vector DNA, the inducingdrug is added to the drinking water of the animals at a near maximaldose (based on manufacturer's recommendation) for a 6-week trial and theexpression is assessed by serum hAAT ELISA. If induction is observed,the drug is removed for 6 weeks to allow for wash-out, and then drug isre-added at one-third the concentration used for the original induction.This process is repeated until the minimum dose required for detectableinduction is determined. If there is no detectable induction at thefirst dose of inducer drug, then the concentration in the drinking wateris tripled for another 6-week trial prior to termination of the study.

6. Polypeptide and Peptide Sequences

6.1 Illustrative ApoE Peptide Sequences of the Present Invention

6.2 Illustrative Mammalian Lipoprotein Receptor Targeting PeptidesSHLRKLRKRLLRD (SEQ ID NO:1) (human ApoE, GenBank #Q28995) SHLRKLRERLLRD(SEQ ID NO:2) (GenBank #1EA8_A ApoE3 peptide) SHLRKMRKRLLRD (SEQ IDNO:3) (tree shrew ApoE, GenBank #AAG21401) SHLRKLPKRLLRD (SEQ ID NO:4)(bovine ApoE, GenBank # S26478) SHLRKLRQRLLRD (SEQ ID NO:5) (fromGenBank 1H7I_A ApoE3 peptide) SHMRKLRKRVLRD (SEQ ID NO:6) (from canineApoE, GenBank # C60940) SHLRKMRKRLMRD (SEQ ID NO:7) (rat ApoE GenBank#NP_033826) SHLRRLRRRLLRD (SEQ ID NO:8) (murine Riken GenBank#XP_233702)

ApoE Consensus Sequence #1: SHX₁RX₂X₃X₄X₅RX₆X₇RD (SEQ ID NO:9)

-   where-   X₁=Leu or Met;-   X₂=Lys or Arg;-   X₃=Leu or Met;-   X₄=Arg or Pro;-   X₅=Lys Glu, Gln or Arg;-   X₆=Leu or Val; and-   X₇=Leu or Met

ApoE Consensus Sequence #2: SHXRXXXXRXXRD (SEQ ID NO:10)

where X=any amino acid LRKLRKRLLR (SEQ ID NO:11) (from GenBank #Q28995)LRKLRERLLR (SEQ ID NO:12) (from GenBank #1EA8_A) LRKMRKRLLR (SEQ IDNO:13) (from GenBank #AAG21401) LRKLPKRLLR (SEQ ID NO:14) (from GenBank# S26478) LRKLRQRLLR (SEQ ID NO:15) (from GenBank 1H7I_A) MRKLRKRVLR(SEQ ID NO:16) (from GenBank # C60940) LRKMRKRLMR (SEQ ID NO:17) (fromGenBank #NP_033826) LRRLRRRLLR (SEQ ID NO:18) (from GenBank #XP_233702)

ApoE Consensus Sequence #3: X₁RX₂X₃X₄X₅RX₆X₇R (SEQ ID NO:19)

-   where-   X=Leu or Met;-   X₂=Lys or Arg;-   X₃=Leu or Met;-   X₄=Arg or Pro;-   X₅=Lys Glu, Gln or Arg;-   X₆=Leu or Val; and-   X₇=Leu or Met

ApoE Consensus Sequence #4: XRXXXXRXXR (SEQ ID NO:20)where X=any amino acid

6.3 Illustrative Lipid-Associated Protein (LAP)-Derived Peptide SequenceDWLKAFYDKVAEDLDEAF (SEQ ID NO:21)

6.4 Illustrative LR Targeting Ligands Comprising ApoE and LAP PeptidesLRKLRKRLLRDWLKAFYDKVAEDLDEAF (SEQ ID NO:22) LRKLRERLLRDWLKAFYDKVAEDLDEAF(SEQ ID NO:23) LRKMRKRLLRDWLKAFYDKVAEDLDEAF (SEQ ID NO:24)LRKLPKRLLRDWLKAFYDKVAEDLDEAF (SEQ ID NO:25) LRKLRQRLLRDWLKAFYDKVAEDLDEAF(SEQ ID NO:26) MRKLRKRVLRDWLKAFYDKVAEDLDEAF (SEQ ID NO:27)LRKMRKRLMRDWLKAFYDKVAEDLDEAF (SEQ ID NO:28) LRRLRRRLLRDWLKAFYDKVAEDLDEAF(SEQ ID NO:29)

ApoE-LAP Consensus Peptide Sequence #5:X₁RX₂X₃X₄X₅RX₆X₇RDWLKAFYDKVAEDLDEAF (SEQ ID NO:30)

-   where-   X₁=Leu or Met;-   X₂=Lys or Arg;-   X₃=Leu or Met;-   X₄=Arg or Pro;-   X₅=Lys Glu, Gln or Arg;-   X₆=Leu or Val; and-   X₇=Leu or Met

ApoE-LAP Consensus Peptide Sequence #6: XRXXXXRXXRDWLKAFYDKVAEDLDEAF(SEQ ID NO:31)where X=any amino acid

7 Illustrative Therapeutic Genes Useful in the Present Invention TABLE14 Growth Factors Factor Principal Source Primary Activity Comments PDGFplatelets, promotes proliferation two different protein endothelialcells, of connective tissue, chains form 3 distinct placenta glial andsmooth dimer forms; AA, AB muscle cells and BB EGF submaxillary gland,promotes proliferation Brunners gland of mesenchymal, glial andepithelial cells TGF-α common in may be important for related to EGFtransformed cells normal wound healing FGF wide range of cells; promotesproliferation at least 19 family protein is of many cells; inhibitsmembers, 4 distinct associated with the some stem cells; receptors ECMinduces mesoderm to form in early embryos NGF promotes neurite severalrelated outgrowth and neural proteins first cell survival identified asprotooncogenes; trkA (trackA), trkB, trkC Erythropoietin kidney promotesproliferation and differentiation of erythrocytes TGF-β activated TH₁cells anti-inflammatory at least 100 different (T-helper) and(suppresses cytokine family members natural killer (NK) production andclass II cells MHC expression), promotes wound healing, inhibitsmacrophage and lymphocyte proliferation IGF-I primarily liver promotesproliferation related to IGF-II and of many cell types proinsulin, alsocalled Somatomedin C IGF-II variety of cells promotes proliferationrelated to IGF-I and of many cell types proinsulin primarily of fetalorigin

TABLE 15 Interleukins and Interferons Interleukins Principal SourcePrimary Activity IL1-α and -β macrophages and other costimulation ofAPCs and T cells, antigen presenting cells inflammation and fever, acutephase (APCs) response, hematopoiesis IL-2 activated TH₁ cells, NKproliferation of B cells and activated T cells cells, NK functions IL-3activated T cells growth of hematopoietic progenitor cells IL-4 TH₂ andmast cells B cell proliferation, eosinophil and mast cell growth andfunction, IgE and class II MHC expression on B cells, inhibition ofmonokine production IL-5 TH₂ and mast cells eosinophil growth andfunction IL-6 activated TH₂ cells, acute phase response, B cellproliferation, APCs, other somatic cells thrombopoiesis, synergisticwith IL-1 and TNF on T cells IL-7 thymic and marrow T and Blymphopoiesis stromal cells IL-8 macrophages, other chemoattractant forneutrophils and T somatic cells cells IL-9 T cells hematopoietic andthymopoietic effects IL-10 activated TH₂ cells, CD8⁺ inhibits cytokineproduction, promotes B T and B cells, cell proliferation and antibodyproduction, macrophages suppresses cellular immunity, mast cell growthIL-11 stromal cells synergisitc hematopoietic and thrombopoietic effectsIL-12 B cells, macrophages proliferation of NK cells, INF- production,promotes cell-mediated immune functions IL-13 TH₂ cells IL-4-likeactivities Interferons Principal Source Primary Activity INF-α and -βmacrophages, neutrophils antiviral effects, induction of class I MHC andsome somatic cells on all somatic cells, activation of NK cells andmacrophages INF-γ activated TH₁ and NK induces of class I MHC on allsomatic cells cells, induces class II MHC on APCs and somatic cells,activates macrophages, neutrophils, NK cells, promotes cell- mediatedimmunity, antiviral effects

8. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference in whole or in part:

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1. An adeno-associated viral vector comprising a first polynucleotide comprising a first nucleic acid segment that encodes an AAV capsid protein that comprises an exogenous amino acid sequence that binds to a mammalian lipoprotein receptor.
 2. The vector of claim 1, wherein said capsid protein is a Vp1 or a Vp2 capsid protein.
 3. The vector of claim 1, wherein said exogenous amino acid sequence binds to a mammalian low-density lipoprotein (LDL) or very low density lipoprotein (VLDL) receptor.
 4. The vector of claim 1, wherein said exogenous amino acid sequence comprises the sequence of any one of SEQ ID NO:1 to SEQ ID NO:21.
 5. (canceled)
 6. The vector of claim 1, wherein said exogenous amino acid sequence comprises the sequence of any one of SEQ ID NO:1 to SEQ ID NO:20, and further comprises the sequence of SEQ ID NO:21.
 7. The vector of claim 1, wherein said exogenous amino acid sequence comprises the sequence of any one of SEQ ID NO:22 to SEQ ID NO:31.
 8. A recombinant adeno-associated viral expression system comprising: (a) a first polynucleotide comprising a first nucleic acid segment that encodes an AAV capsid protein that comprises an exogenous amino acid sequence that binds to a mammalian lipoprotein receptor; and (b) a second polynucleotide comprising a second nucleic acid segment that encodes an expressed therapeutic agent.
 9. (canceled)
 10. The recombinant adeno-associated viral expression system of claim 8, wherein said exogenous amino acid sequence binds to a mammalian VLDL or LDL receptor.
 11. The recombinant adeno-associated viral expression system of claim 8, wherein said exogenous amino acid sequence comprises the sequence of any one of SEQ ID NO:1 to SEQ ID NO:20.
 12. The recombinant adeno-associated viral expression system of claim 11, wherein said exogenous amino acid sequence further comprises the sequence of SEQ ID NO:21.
 13. The recombinant adeno-associated viral expression system of claim 8, wherein said exogenous amino acid sequence comprises the sequence of any one of SEQ ID NO:22 to SEQ ID NO:31.
 14. The recombinant adeno-associated viral expression system of claim 8, wherein said first and said second polynucleotides are comprised within a single rAAV vector:
 15. The recombinant adeno-associated viral expression system of claim 8, wherein said first and said second polynucleotides are comprised on distinct rAAV vectors:
 16. The recombinant adeno-associated viral expression system of claim 8, wherein said second polynucleotide further comprises a promoter operably linked to said second nucleic acid segment, wherein said promoter expresses said therapeutic agent.
 17. (canceled)
 18. (canceled)
 19. The recombinant adeno-associated viral expression system of claim 16, wherein said promoter is a mammalian β-actin promoter.
 20. The recombinant adeno-associated viral expression system of claim 8, wherein said second polynucleotide further comprises an enhancer sequence operably linked to said second nucleic acid segment.
 21. (canceled)
 22. The recombinant adeno-associated viral expression system of claim 20, wherein said enhancer sequence comprises a CMV enhancer.
 23. The recombinant adeno-associated viral expression system of claim 8, wherein said second nucleic acid segment further comprises a post-transcriptional regulatory sequence.
 24. The recombinant adeno-associated viral expression system of claim 23, wherein said regulatory sequence comprises a woodchuck hepatitis virus post-transcription regulatory element. 25.-27. (canceled)
 28. The recombinant adeno-associated viral expression system of claim 8, wherein said therapeutic agent is an α₁-antitrypsin (AAT) polypeptide.
 29. (canceled)
 30. A recombinant adeno-associated virus virion comprising the vector of claim 1, or the recombinant adeno-associated viral expression system of claim
 8. 31. (canceled)
 32. A plurality of adeno-associated viral particles comprising the vector of claim 1 or the recombinant adeno-associated viral expression system of claim
 8. 33. A mammalian cell comprising the vector of claim 1, or the recombinant adeno-associated viral expression system of claim
 8. 34.-42. (canceled)
 43. A kit comprising: (a) the adeno-associated viral vector of claim 1, or the recombinant adeno-associated viral expression system of claim 8; and (b) instructions for using said kit.
 44. A method for targeting an AAV virion or viral particle to a mammalian cell that comprises a cell-surface lipoprotein receptor, said method comprising the step of: providing to a population of cells an AAV virion or viral particle that comprises the vector of claim 1, or the recombinant adeno-associated viral expression system of claim 8, in an amount and for a time effective to target said virion or said viral particle to cells of said population that express said cell-surface lipoprotein receptor.
 45. A method for targeting an expressed therapeutic agent to a mammalian cell that comprises a cell-surface lipoprotein receptor, said method comprising the step of providing to a mammal that comprises a population of said cells an amount of the recombinant adeno-associated viral expression system of claim
 8. 46. (canceled)
 47. A method for preventing, treating or ameliorating the symptoms of a disease, dysfuinction, or deficiency in a mammal, said method comprising administering to said mammal the virion of claim 30, or the plurality of adeno-associated viral particles of claim 32, in an amount and for a time sufficient to treat or ameliorate the symptoms of said disease, dysfunction, or deficiency in said mammal. 48.-51. (canceled) 